Neurofibrillary labels

ABSTRACT

Disclosed are methods for determining the stage of neurofibrillary degeneration associated with a tauopathy in a subject believed to suffer from the disease, which methods comprise the steps of: (i) introducing into the subject a ligand capable of labelling aggregated paired helical filament (PHF) tau protein, (ii) determining the presence and\or amount of ligand bound to extracellular aggregated PHF tau in the medial temporal lobe of the brain of the subject, (iii) correlating the result of the determination made in (ii) with the extent of neurofibrillary degeneration in the subject. The methods can be used for pre-mortem diagnosis and staging of tauopathies such as Alzheimer&#39;s Disease. Preferred ligands include sulphonated-benzothiazole-like compounds and diaminophenothiazines. Novel ligands (e.g. sulphonated-benzothiazole-like compounds) are also provided. The method may also include the use of “blocking ligands” to block competing binding sites. In other aspects the invention provides in vitro methods for identifying ligands capable of labeling aggregated PHF tau protein, the methods comprising the steps of: (i) providing a first agent suspected of being capable of labeling aggregated PHF tau protein, (ii) contacting (a) a tau protein or a derivative thereof containing the tau core fragment bound to a solid phase so as to expose a high affinity tau capture site, with (b) a liquid phase tau protein or derivative thereof capable of binding to the solid phase tau protein or derivative, and (c) said selected first agent and (d) a second agent known to be tau-tau binding inhibitor, (iii) selecting first agent which fully or partially relieves the inhibition of binding of the liquid phase tau protein or derivative of (b) to the solid phase tau protein or derivative of (a) by the inhibitor (d). Ligands may also be tested to confirm that they are not themselves inhibitors.

FIELD OF THE INVENTION

The present invention concerns materials, methods and models relatinggenerally to the labelling and detection of neurofibrillary tangles. Inaddition, it concerns the identification and development of ligandssuitable for neuropathological staging and their use in the diagnosis,prognosis or treatment of diseases such as Alzheimer's Disease (AD).

BACKGROUND TO THE INVENTION Neuropathological Staging and AD

The neuropathological staging proposed by Braak (Braak, H et al. (1991),Acta. Neuropathol. 82, 239-259) provides the best available definitionof progression of relatively pure neurofibrillary degeneration of theAlzheimer-type which is diagnostic of AD (Wischik et al. (2000),“Neurobiology of Alzheimer's Disease”, Eds. Dawbarn et al., TheMolecular and Cellular Neurobiology Series, Bios Scientific Publishers,Oxford). This staging is shown schematically in terms of brain region inFIG. 2B, and is based on a regular regional hierarchy of neurofibrillarytangle (NFT) distribution. Regions of the brain which appear earlier inthe hierarchy have both more tangles and are affected in less severecases than those later in the list.

Relationship Between AD, Clinical Dementia and Neuropathological Staging

The provision of an effective pre-mortem assessment of Braak Stage wouldbe of use in the assessment and treatment of AD, for which thedifferential includes Lewy Body dementia, Parkinson's disease, variousforms of fronto-temporal and cortico-basal degeneration, progressivesupranuclear palsy and a range of rare neurological syndromes.

The original model proposed by Braak was essentially qualitative innature, and was not linked to any implications about the threshold fordevelopment of clinical dementia and symptoms.

In terms of the appearance of clinical dementia by DSM-IV criteria, thiscorresponds statistically to the transition between Braak stages 3 and 4(FIG. 2 c). The DSM-IV criteria (Diagnostic and Statistical Manual ofMental Disorders, 4^(th) Edition, American Psychiatric Association,American Psychiatric Press, Washington D.C. (1994)) for the definitionof dementia equate to an MMSE (Mini-Mental State Examination) cut-offpoint of about 18, and corresponds to a dementia prevalence of about 5%of the population over-65 years old (over-65's represent about 17% ofthe total population).

Gertz et al. ((1996) Eur. Arch. Psychiatry Clin. Neurosci. 246, 132-6))studied cases followed from general practice to post-mortem, which wererigorously characterised at the clinical level using CAMDEX (Roth et al,1988, “The Cambridge Examination for Mental Disorders of the Elderly(CAMDEX)” Cambridge University Press). These were staged post-mortem bythe criteria of Braak and, after excluding all cases with any degree ofvascular pathology found post-mortem, there remained uncertainty inabout one third of cases at the point of transition. That is, about onethird of cases with a clinical diagnosis of AD are actually at earlyBraak stages (stages 1-3), have vascular pathology, or have concomitantLewy body pathology. Thus there exists a high degree of uncertainty,even in the best practice research setting. The predominantneuropathological substrate that is actually present when a routineclinical diagnosis of AD is made is even more uncertain.

It has recently been reported that a molecule (FDDNP,2-(1-{6-[(2-[¹⁸F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile)demonstrates increased relative retention time (RRT) in medial temporallobe brain regions (hippocampus, entorhinal cortex and amygdala)following injection and PET imaging in cases with a clinical andneuroradiological diagnosis of Alzheimer's disease (Shoghi-Jadid et al.,Am. J. Geriatr. Psychiatr. 2002, 10:24-35).

Although binding to NFTs and amyloid plaques is discussed, no binding toNFTs is shown, although the compound does bind with high affinity tosynthetic beta-amyloid fibrils in vitro.

When cases were matched for corresponding disease severity by MMSE scorewith a neuropathological case series in which vascular pathology wasexcluded, the RRT values reported by Shoghi-Jadid et al. were found tocorrelate with beta-amyloid plaque counts but not with measures ofneurofibrillary tangle pathology, as shown below.

Spearman Rank Correlation Coefficients:

MTL AP Global AP MTL NFT Global NFT RRT 0.665** 0.654** 0.244 0.189 p<0.01 <0.01 >0.1 >0.1

Pearson Correlation Coefficients:

MTL AP Global AP MTL NFT Global NFT RRT 0.602* 0.596* 0.266 0.275 p<0.05 <0.05 >0.3 >0.3

Where the Parameters are Defined as Follows:

MTL AP medial temporal lobe amyloid plaques Global AP average amyloidplaque load in 12 brain regions MTLNFT medial temporal lobeneurofibrillary tangles Global NFT average NFT load in 12 brain regions

However, beta-amyloid deposition is known to discriminate poorly betweennormal aging and Alzheimer's disease (see FIG. 2 d herein), andbeta-amyloid pathology does not provide a sound basis forneuropathological staging (Braak and Braak, 1991). Therefore, FDDNP-RRTdoes not provide a method for in vivo neuropathological staging ofAlzheimer's disease.

Although it is possible that further refinement in clinical methods withparticular reference to more specific neuropsychological indicators(e.g. split attention tasks, delayed matching to sample, etc.) mayimprove the accuracy of clinical diagnosis, an essential problem is todevelop methods for the direct measurement of underlying neuropathologyduring life, in particular the extent of neurofibrillary degeneration ofthe Alzheimer-type.

Progression of Neurofibrillary Degeneration and Tau

As described above, the tau-based pathology of AD is a major feature ofthe phenotype. It is highly correlated with extent of neuronaldestruction (reviewed in Wischik et al. (2000) loc cit).

On a cellular basis, the formation of NFTs from Tau is believed toproceed as follows. In the course of their formation and accumulation,paired helical filaments (PHFs) first assemble as filaments within thecytoplasm, probably from early tau oligomers which become truncatedprior to, and in the course of, PHF assembly (Refs 26,27). They then goon to form classical intracellular NFTs. In this state, PHFs consist ofa core of truncated tau and a fuzzy outer coat containing full-lengthtau (Wischik et al. (2000) loc. cit.). The assembly process isexponential, consuming the cellular pool of normal functional tau andinducing new tau synthesis to make up the deficit (Ref 29). Eventuallyfunctional impairment of the neurone progresses to the point of celldeath, leaving behind an extracellular NFT. Cell death is highlycorrelated with the number of extracellular NFTs (Bondareff, W. et al.(1993) Arc. Gen. Psychiatry 50: 350-6). As the outer neuronal membraneis damaged and NFTs are extruded into the extracellular space, there isprogressive loss of the fuzzy outer coat of the neurone withcorresponding loss of N-terminal tau immunoreactivity, but preservationof tau immunoreactivity associated with the PHF core (FIG. 3; Ref 30).

In the process of aggregation, tau protein undergoes a conformationalchange in the repeat domain associated with a half-repeat phase-shift(Refs 32,33). This creates a proteolytically-stable fragment which isidentical to that found in the core of the paired helical filaments(PHFs) which constitute the neurofibrillary tangles characteristic ofAD. By analogy with other protein aggregation systems, the process mostlikely involves an alpha-helix to beta-strand change in conformation(reviewed in Wischik et al. (2000) loc. cit.).

Generally speaking therefore, the aggregation of tau can be consideredin 3 stages: intracellular oligomers; intracellular filaments (stage 1of FIG. 3); extracellular filaments (stages 2 & 3 of FIG. 3).

However, to date, no definitive correlation has been established betweenthese stages, which occur at the cellular level, and possibly atdifferent rates and probabilities in different regions in the brain, andthe progression of pathology according to the defined hierarchicalsystem of Braak and Braak which, as discussed above, is the bestavailable definition of progression of relatively pure neurofibrillarydegeneration.

Invasive Methods for Assessing AD

Lumbar-puncture CSF measurements enable discrimination between AD andcontrols, and between AD and other neurological disorders, butlumbar-puncture is more invasive then nuclear medicine-based approaches,and carries a higher risk (Refs. 17 to 21). EEG-neurological diagnosishas also been developed (Refs 22-25), but in this regard there remains aneed for cheap instrumentation which can be used at the point ofclinician contact.

Neurofibrillary Degeneration Via Brain Atrophy—SPECT and PET

Numerous studies have shown that global brain atrophy and specificmedial temporal lobe atrophy, particularly of the hippocampus, areclosely linked to underlying neurofibrillary degeneration of theAlzheimer-type, and are valuable in the early diagnosis of AD (Refs1-8).

However, although the diagnosis of AD by monitoring global brain atrophyrepresents a methodology which can be made to work in a researchsetting, there are difficulties in defining and measuring atrophy inspecific brain regions, and likewise in the measurement of globalneocortical atrophy. In any case, a diagnosis based on detectableatrophy may come too late for effective treatment.

There have been advances in diagnostic methodology in recent years,following the identification of diagnostic features in SPECT scans (Refs9-12; characteristic patterns of perfusion defect detected by HMPAOSPECT), PET scans (Refs 13-15; metabolic defect detected by glucosemetabolism profile) and MRI scans (Ref 16; global brain atrophy,specific patterns of lobar atrophy). Of these, the most generallyaccessible are MRI and SPECT, since PET is for the present time limitedto centres which have local specialised cyclotron and radiochemistrycapability for the preparation of short half-life injectableradioligands (Aberdeen, London, Cambridge in UK). Notably, thecharacteristic early stage temporo-parietal perfusion defect detected byHMPAO SPECT in patients with AD corresponds very closely to the patternof tau pathology which can be detected biochemically (FIG. 1). Thebiochemical changes precede overt neurofibrillary degeneration as seenby the appearance of NFTs (FIG. 2; Mukaetova-Ladinska et al., 2000 Am.J. Pathol. Vol. 157, No. 2, 623-636).

However, although MRI and SPECT scans are useful for detecting specificpatterns of perfusion defects characteristic of AD, discriminationbetween different neuropathological stages, or between AD and othertypes of dementia, is difficult.

For instance, SPECT is useful for detection of a specific pattern ofbilateral temporo-parietal perfusion defect that is characteristic of AD(Refs 9-11), which can be useful even at very early stage disease.However, SPECT changes discriminate neuropathological stages poorly (Ref12). Furthermore, discrimination between AD and Lewy Body dementia isdifficult. Both have a bilateral temporo-parietal perfusion defect, butonly in the latter does an occipital perfusion defect tend to bepresent. The same patterns of defect can be demonstrated using PETmeasurement of glucose metabolism (Refs 13-15), but the problem ofdistinguishing Lewy Body dementia persists in this modality.

Thus, as can be inferred from data in Ref 12, the probability ofsuccessful SPECT detection of cases at Braak stages 1&2 is 50%, and atstages 3&4 is 60%. It is only at stages 5&6 that 95% of cases becomeSPECT-positive. Conversely, cases detected as SPECT-positive could be atstages 1&2 (20%), 3&4 (20%), or 5&6 (60%). SPECT will therefore fail todetect 40-50% of the pre-stage 4 target population for early diagnosisand therapeutic intervention. In a further study (data not shown) it wasshown that overall agreement between SPECT diagnosis and clinicaldiagnosis was of the order of 50%.

Thus, in developing a treatment aimed specifically at preventingneurofibrillary degeneration of the Alzheimer-type, there is a criticalneed to develop, in parallel, non-invasive means of selecting patientsfor treatment, and monitoring their response to the treatment, accordingto a defined and reproducible definition of disease progression.

DISCLOSURE OF THE INVENTION Brief Summary of the Invention

The present inventors have used immunochemical properties (Refs 26, 27,30) to distinguish intracellular tangles from extracellular tangles.Both the frequency of cases with tangles in these categories (i.e.probability) and their quantity (i.e. counts per mm²) were eredetermined in a prospective case series and grouped into the regions ofthe brain known to represent stages in the progression of pathologyaccording to the system of Braak and Braak.

As described in more detail below, these antibody studies demonstratefor the first time that by employing extracellular vs. intracellularspecificity, in defined brain regions, deposits of PHF-tau provide abasis for empirical staging of the neurofibrillary degeneration of AD.

Thus in one aspect, the present invention provides a method fordetermining the stage of neurofibrillary degeneration associated withtauopathy (e.g. AD) in a subject believed to suffer from the disease,which method comprises the steps of:

(i) introducing into the subject a ligand capable of labellingaggregated PHF tau,(ii) determining the presence and\or amount of ligand bound toextracellular aggregated PHF tau in the medial temporal lobe of thebrain of the subject,(iii) correlating the result of the determination made in (ii) with theextent of neurofibrillary degeneration in the subject.

As described in the introduction, progression of neurofibrillarydegeneration is definitive of the neuropathological staging proposed byBraak, which in turn is the best available neuropathological definitionof progression of AD. The methods of the present invention can thus beused to provide an actual Braak stage result. In preferred embodimentsthey can be used to diagnose patients at early Braak stages (e.g. Braakstage 2) even before clinical symptoms may be readily apparent—suchdiagnosis can be used to direct timely treatment and advice.

Interestingly, results shown in Gertz et al. (1996) loc cit, based onimmunological detection of NFTs, but which did not distinguishextracellular and intracellular tangles, showed little differencebetween the numbers detected in demented (generally Braak stage 4-6) andnon-demented (generally Braak stage 1-3) subjects in medial temporallobe structures (see FIG. 1 and Table 2, page 134 therein; the relevantstructures are labelled Pre alpha ent., CA1, Pri Ento.). Thus thecorrelation demonstrated by the present invention is particularlysurprising.

The invention further provides novel ligands for use in labelling tauaggregates, plus also novel screens for discovering such ligands.

Some of the aspects of the invention discussed above will now be dealtwith in more detail.

Choice of Subject

Suitable subjects for the method may be selected on the basis ofconventional factors. Thus the initial selection of a patient mayinvolve any one or more of: rigorous evaluation by experiencedclinician; exclusion of non-AD diagnosis as far as possible bysupplementary laboratory and other investigations; objective evaluationof level of cognitive function using neuropathologically validatedbattery.

Ligands

The ligand is capable of labelling aggregated PHF tau, the formation ofwhich is discussed above. It may specifically or preferentially bindsuch tau (preferentially with respect to competing binding sites presentin the relevant region of the brain). Suitable ligands (including novelligands) and methods of identifying them are discussed below.

More specifically, the disclosure that Braak staging can be assessed onthe basis described herein has significant implications for the choiceand\or development of ligands for use in diagnostic labelling.Immunological methods suffer from the drawback that antibodies do notreadily cross the blood-brain barrier in a quantitative manner, andfurthermore, the method may be clinically unsuitable since adversereactions may be triggered by the injection of antibodies into the bodyfor this purpose. It is consequently difficult to discriminate betweendifferent stages of tau aggregation on the basis of differentialpatterns of immunoreactivity in living subjects.

The present inventors have therefore investigated the critical chemicalcharacteristics of compounds which bind to neurofibrillary tangles. Theyprovide herein a minimal chemical structure required for binding whichhas implications, inter alia, in the development and use of compounds asligands of neurofibrillary tangles and such processes, uses andcompounds form further aspects of the invention.

Preferred ligands, including novel ligands, are disclosed in more detailhereinafter, but may include in particularsulphonated-benzothiazole-like compounds (see e.g. FIG. 4 a) anddiaminophenothiazines (see e.g. FIG. 8) as well as other mimeticcompounds sharing an appropriate minimal chemical structure with eitherof these. Compositions comprising, or consisting of, combinations of theligands disclosed herein (preferably distinguishable ligands e.g. interms of labelling) and\or combinations of ligands with blocking agents(see below) form various aspects of the invention.

Binding to Extracellular Tau

The determination of (ii) above is made based on extracellularaggregated tau. In general terms, for the purposes of the presentinvention, this may be determined from extracellular tangles (see e.g.Refs 26, 27 and Examples, Methods and Materials, Table).

It has previously been shown from histological studies that, during thecourse of aggregation, tau protein acquires binding sites for compoundssuch as thiazin red and thioflavin-S (Refs 26, 27). The binding site canbe shown to exist within the tangle itself, and not in extraneousproteins (Ref 34). Thus both intracellular and extracellular tangles arelabelled to some extent by such ligands, as judged histologically.

In general terms, the probability or amount of extracellular bindingsites (as opposed to total binding sites, or intracellular sites) may bedetermined either by using ligands which are too large to gain readyintracellular access, or ligands which can act intracellularly, but at adefined (relatively lower) concentration at which extracellular actionis favoured.

Large chelated ligands, such as those susceptible to detection by SPECT,could be expected to at least reach and bind appropriate extracellulartargets. Compounds labelled directly for PET could potentially detectboth intracellular or extracellular targets, with the latter beingfavoured at low concentration. Thus the work of the present inventorsshows that both of these detection methods have potential in Braakstaging, when used with an appropriate tangle-binding ligand.Nevertheless, in the light of the present disclosure, it will beappreciated that in order to conveniently assess Braak stage via NFTnumbers it may be important to employ ligands which are not only capableof crossing the blood brain barrier and labelling specifiedextracellular or intracellular deposits of aggregated tau, butpreferably can also retain this property when conjugated to furthercompounds.

However, for the avoidance of doubt, ligands may be visualised ordetected by any suitable means, and the skilled person will appreciatethat any suitable detection means as is known in the art could besubstituted for these examples.

Enhancement of Preferential Tau Binding

In one embodiment of the invention, steps (i) and\or (ii) of the methodare performed in conjunction with (preferably preceded by) the furtherstep of introducing into the subject a second ligand which labelscompeting (i.e. non-aggregated tau) binding sites present in therelevant region of the brain preferentially to the first ligand.

Thus the methods and other embodiments herein may include a step:

(ibis) introducing into the subject a blocking ligand which labelsnon-aggregated tau binding sites in the brain of the subjectpreferentially to the ligand capable of labelling aggregated PHF tau.

A competing binding site may be one which is provided by e.g. amyloidplaques, such as may be present in the subject. By introducing suchsecond ligands into the subject, the relative or effective concentrationof first ligand available to bind to aggregated tau may be enhanced.Suitable second ligands (or blocking compounds as they may be describedherein) are described below, but they may in particular includebenzthiazoles such as are shown in FIG. 5, compounds 1B and 2. Anothersuitable blocking ligand may be FDDNP of Shoghi-Jadid et al., Am. J.Geriatr. Psychiatr. 2002, 10:24-35, discussed above.

Brain Regions

The significance of the medial temporal lobe i.e. E2/Trans (Entorhinalcortex layer 2/transitional entorhinal cortex) and E4/HC (Entorhinalcortex layer 4 and hippocampus) regions, and also neocortical structures(F/T/P regions—frontal, temporal, parietal) of the brain aredemonstrated in FIGS. 25, 27, and 29.

In one embodiment, the method comprises only analysing the data based onextracellular NFTs in the medial temporal lobe.

If a further embodiment, both this region and the neocortical structuredata is assessed. In the latter case it may be preferable to assessintracellular PHF deposits.

Thus the methods and other embodiments herein may include a furtherstep:

(iib) additionally determining the presence and\or amount of ligandbound to intracellular aggregated PHF tau in a neocortical structure ofthe brain of the subject,

This may be followed by:

(iii) correlating the result of the determination made in (ii) andoptionally (iib) with the extent of neurofibrillary degeneration in thesubject, and hence the AD state of the subject.

The ligand used for intracellular labelling may in principle be the sameas that used for extracellular labelling, but preferably will bedifferent and\or labelled distinctively (such that it can bedistinguished by whatever imaging process is being used).

The additional steps may be particularly preferred for assessing orconfirming neurofibrillary degeneration in subjects at Braak stage 2-6.

Determination of Neurofibrillary Degeneration

The determination may be of the presence of binding in a given area.This determination can then be related to a normal range of values forcases without any pathology (i.e. cases putatively at Braak Stage 1), orranges of reference values which have been determined for successiveBraak stages to determine the neuropathological stage corresponding tothe given determination. The correlation may be done by means of alook-up table or graph, e.g. based on data corresponding to the Figuresand Table 1 in Example 1 herein for density. Alternatively, a givendetermination may be related with reference to a given threshold valueto a probability of a case being at a Stage more advanced that Stage 1(e.g. based on data corresponding to the Figures herein forprobability), and thereby giving a probability of correctly attributinga diagnosis of Alzheimer's disease.

Uses of the Method

The determination may be as part of a method of diagnosis or prognosis.It may be used to select a patient for treatment, or to assess theeffectiveness of a treatment or a therapeutic e.g. an inhibitor oftau-tau association administered to the subject.

Thus embodiments of the invention include:

A ligand which is capable of labelling extracellular aggregated PHF taufor use in a method of diagnosis or prognosis of AD in a subjectbelieved to suffer from the disease, which method comprises the stepsof:

(i) introducing into the subject a ligand capable of labellingaggregated PHF tau,(ii) determining the presence and\or amount of ligand bound toextracellular aggregated PHF tau in the of the medial temporal lobe ofthe brain of the subject,(iii) correlating the result of the determination made in (ii) with theextent of neurofibrillary degeneration in the subject, and hence the ADstate of the subject.

Use of a ligand which is capable of labelling extracellular aggregatedPHF tau in a method for preparing a diagnostic or prognostic reagentsuitable for use in a method of determining the stage of neurofibrillarydegeneration associated with AD in a subject believed to suffer from thedisease, which method comprises the steps of:

(i) introducing into the subject said reagent which is capable oflabelling aggregated PHF tau,(ii) determining the presence and\or amount of the reagent bound toextracellular aggregated PHF tau in the medial temporal lobe of thebrain of the subject,(iii) correlating the result of the determination made in (ii) with theextent of neurofibrillary degeneration in the subject.

In a still further aspect, the invention provides a kit for performingthe uses and methods described above, the kit comprising one or moreligands or derivatives as provided herein, which are capable of bindingto the aggregated molecules. It may include means for increasing thedetectability of such compounds e.g. a technetium chelating group, plusoptionally means to conjugate this to the ligand, plus optionallytechnetium. Where the kit comprises a derivative of a compound asdisclosed herein, this may be e.g. fluoroscopically detectable, asdiscussed elsewhere in this description. The kit may also include meansfor detecting or visualising the ligand, e.g. where the ligand has anincorporated biotin group, the kit preferably includes an antibiotinantibody. Similarly, the kit may include means for detecting theinherent fluorescence of a compound, means for detectingphotoactivatable groups, further labelled antibodies, etc.

Various preferred ligands for use in the methods and other embodimentsof the present invention will now be discussed in more detail. In eachcase, those skilled in the art will appreciate that instead ofadministering ligands directly, they could be administered in aprecursor form, for conversion to the active form by an activating agentpresent in, or administered to, the same subject.

Sulphonated-Benzothiazole-Like Ligands

Suitable ligands for use in this aspect of the present invention arecompounds of the formula:

wherein:

W is S, O, or NH;

exactly one of X, Y, and Z is CH or N;the others of X, Y, and Z are CH;M¹ is an alkali metal cation;RL is a rigid linker group;Ar¹ is an C₅₋₂₀aryl group;n is an integer from 0 to 3; and,each R^(BT) is a core substituent.

In one embodiment, each of X, Y, and Z is CH, and the compounds have thefollowing formula:

In one embodiment, X is N; Y and Z are each CH; and the compounds havethe following formula:

In one embodiment, Y is N; X and Z are each CH; and the compounds havethe following formula:

In one embodiment, Z is N; X and Y are each CH; and the compounds havethe following formula:

In one embodiment, W is S, and the compounds have the following formula:

In one embodiment, W is O, and the compounds have the following formula:

In one embodiment, W is NH, and the compounds have the followingformula:

In one embodiment, each of X, Y, and Z is CH; and W is S.

In one embodiment, each of X, Y, and Z is CH; and W is O.

In one embodiment, each of X, Y, and Z is CH; and W is NH.

In one embodiment, X is N; Y and Z are each CH; and W is S.

In one embodiment, X is N; Y and Z are each CH; and W is O.

In one embodiment, X is N; Y and Z are each CH; and W is NH.

In one embodiment, Y is N; X and Z are each CH; and W is S.

In one embodiment, Y is N; X and Z are each CH; and W is O.

In one embodiment, Y is N; X and Z are each CH; and W is NH.

In one embodiment, Z is N; X and Y are each CH; and W is S.

In one embodiment, Z is N; X and Y are each CH; and W is O.

In one embodiment, Z is N; X and Y are each CH; and W is NH.

The bicyclic group, comprising W, X, Y, and Z, may be denoted the “coregroup.” When each of X, Y, and Z is CH, and W is S, the compound may bereferred to as a benzothiazole compound, and may be considered to have,as a core group, a benzothiazole group. The “core substituents” may bethen be referred to as “benzothiazole substituents.”

A preferred ligand for use in this aspect of the present invention is aligand compound of the formula (I):

wherein:M¹ is an alkali metal cation;RL is a rigid linker group;Ar¹ is an C₅₋₂₀aryl group;n is an integer from 0 to 3; and,each R^(BT) is a independently benzothiazole substituent.

Both the rigid linker group, RL, and the aryl group, Ar¹, aresubstantially planar. In addition, the rigid linker group, RL, and thearyl group, Ar¹, together with the core group (e.g., benzothiazolegroup), form a compound which is substantially planar. By “substantiallyplanar,” it is meant that the moiety/compound has a high degree ofplanarity e.g. less than 5, 4, 3, 2 or 1° twist between the components,as quantified using standard chemical models and assumptions. Preferablythe twist will be no greater than that of the compound of FIG. 16.

In one embodiment, the compound has a compound length which is fromabout 14.7 AU to about 15.3 AU.

The present inventors have determined that compounds having thecharacteristics described above may be particularly suitable for the‘Braak staging’ methods of the invention. Such compounds may be known inthe art, or may be novel as described in more detail below.

The “compound length” is the distance between the two most distantaromatic ring atoms (denoted “reference atoms”). For example, forbenzothiazole compounds, at the benzothiazole “end” of the molecule, thereference atom will be one of two atoms:

At the aryl “end” of the molecule, when Ar¹ is an aryl group having aphenyl core (see below), the reference atom will be one of three atoms:

Distances used herein may be computed using ‘Chemical Database Service’,Daresbury, and the Cambridge Structure Database, using ‘Chemicalstructure search and retrieval software’. This data and software areavailable in the public domain.

In one embodiment, M is Li, Na, K, or Cs.

In one embodiment, M is Na or K.

In one embodiment, n is 0. In one embodiment, n is 1.

In one embodiment, n is 2. In one embodiment, n is 3.

In one embodiment, each R^(BI)' is independently selected from:

C₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, and amino.

In one embodiment, each R^(BT) is independently selected from: -Me, -Et,-nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl, —Br,—I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂, N(iPr)₂,and —N(nPr)₂.

In one embodiment, each R^(BT) is independently selected from:C₁₋₄alkyl. In one embodiment, each R^(BT) is selected from: -Me, -Et,-nPr, and -iPr. In one embodiment, each R^(BT) is -Me.

In one embodiment, n is 1 and R^(BT) is -Me, -Et, -nPr, or -iPr.

In one embodiment, n is 1 and R^(BT) is -Me.

In one embodiment, the compound has the following formula:

In one embodiment, the compound has the following formula:

In one embodiment, RL is a group of the formula:

wherein m is an integer from 0 to 4, and each R^(RL) is independently arigid linker aryl substituent, and the compounds have the formula:

In one embodiment, m is 0. In one embodiment, m is 1.

In one embodiment, m is 2. In one embodiment, m is 3.

In one embodiment, m is 4.

In one embodiment, each R^(RL) is independently selected from:

C₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, and amino.

In one embodiment, each R^(RL) is independently selected from: -Me, -Et,-nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl, —Br,—I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂, N(iPr)₂,and —N(nPr)₂.

In one embodiment, each R^(RL) is independently selected from:C₁₋₄alkyl.

In one embodiment, RL is a group of the formula:

In one embodiment, RL is a group of the formula:

wherein p is an integer from 0 to 3, and each R^(RL) is independently arigid linker aryl substituent, and the compounds have the formula:

In one embodiment, p is 0. In one embodiment, p is 1.

In one embodiment, p is 2. In one embodiment, p is 3.

In one embodiment, each R^(RL) is independently selected from:

C₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, and amino.

In one embodiment, each R^(RL) is independently selected from: -Me, -Et,-nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl, —Br,—I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂, N(iPr)₂,and —N(nPr)₂.

In one embodiment, each R^(RL) is independently selected from:C₁₋₄alkyl.

In one embodiment, RL is a group of the formula:

The aryl group, Ar¹, is a C₅₋₂₀aryl group. The term “C₅₋₂₀aryl,” as usedherein, pertains to a monovalent moiety obtained by removing a hydrogenatom from an aromatic ring atom of a C₅₋₂₀aromatic compound, saidcompound having one ring, or two or more rings (e.g., fused), and havingfrom 5 to 20 ring atoms, and wherein at least one of said ring(s) is anaromatic ring. Preferably, each ring has from 5 to 7 ring atoms. “C₅₋₂₀”denotes ring atoms, whether carbon atoms or heteroatoms.

Examples of C₅₋₂₀aryl groups which do not have ring heteroatoms (i.e.,C₅₋₂₀carboaryl groups) include, but are not limited to, those derivedfrom benzene (i.e., phenyl) (C₆), naphthalene (C₁₀), anthracene (C₁₄),phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene (C₁₆).

Examples of C₅₋₂₀heteroaryl groups include, but are not limited to,C₅heteroaryl groups derived from furan (oxole), thiophene (thiole),pyrrole (azole), imidazole (1,3-diazole), pyrazole (1,2-diazole),triazole, oxazole, isoxazole, thiazole, isothiazole, oxadiazole, andoxatriazole; and C₆heteroaryl groups derived from isoxazine, pyridine(azine), pyridazine (1,2-diazine), pyrimidine (1,3-diazine; e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine), triazine, tetrazole,and oxadiazole (furazan).

Examples of C₅₋₂₀heterocyclic groups (including C₅₋₂₀heteroaryl groups)which comprise fused rings, include, but are not limited to,C₉heterocyclic groups derived from benzofuran, isobenzofuran, indole,isoindole, purine (e.g., adenine, guanine), benzimidazole;C₁₀heterocyclic groups derived from quinoline, isoquinoline,benzodiazine, pyridopyridine, quinoxaline; C₁₃heterocyclic groupsderived from carbazole; C₁₄heterocyclic groups derived from acridine,xanthene, phenoxathiin, phenazine, phenoxazine, phenothiazine.

In one embodiment, Ar¹ is an aryl group having a phenyl core, and hasthe formula:

wherein q is an integer from 0 to 5; and each R^(A) is independently anaryl substituent; R^(C), is present, is a reactive conjugatingsubstituent, or R^(C) is, or contains, a detectable label; and thecompound has the formula:

In one embodiment, R^(C), if present, is a reactive conjugatingsubstituent, and is a group which is suitable for conjugation to anothermolecule or chemical species.

In one embodiment, R^(C), if present, is a reactive conjugatingsubstituent, and is, or contains, a reactive functional group suitablefor conjugation to another molecule by chemical reaction therewith, toform a covalent linkage therebetween. Examples of suitable reactivefunctional groups include active esters (e.g., succinimidyl esters).

In one embodiment, R^(C), if present, is a reactive conjugatingsubstituent, and is, or contains, a moiety suitable for conjugation toanother molecule by a strong non-covalent interaction. Examples of suchgroups include biotin (for binding with molecules bearing avidin orstreptavidin).

In one embodiment, R^(C), if present, is a reactive conjugatingsubstituent, and is, or contains, a moiety suitable for conjugation toanother molecule by complex or chelate formation, e.g., a chelatinggroup. Examples of such groups include groups which complex with orchelate, e.g., metal ions, e.g., technetium ions. Examples of suchgroups include diethylenetriaminepentaacetic acid.

In one embodiment, R^(C), if present, is, or contains, a detectablelabel. Examples of detectable labels include, e.g., dyes, fluorescentmarkers, antigenic groups, stable and unstable isotopes, andpositron-emitting carbon atoms. In one embodiment, R^(C), if present,is, or contains, a detectable label comprising a stable isotope. In oneembodiment, R^(C), if present, is, or contains, a detectable labelcomprising an unstable isotope. In one embodiment, R^(C), if present,is, or contains, ¹⁸F. In one embodiment, R^(C), if present, is, orcontains, a detectable label comprising a positron-emitting carbon atom.

Further R^(C) substituents are discussed below.

In one embodiment, R^(C) is present, and is as defined above.

In one embodiment, q is 0. In one embodiment, q is 1.

In one embodiment, q is 2. In one embodiment, q is 3.

In one embodiment, q is 4. In one embodiment, q is 5.

In one embodiment, each R^(A) is independently selected from: —OH, —NH₂,—NHR¹, —NR¹R², —SO₃M², C₁₋₄alkyl, wherein R¹ and R² are each C₁₋₄alkyl,and M² is an alkali metal cation, as defined above.

In one embodiment, at least one R^(A) is —OH or —NH₂.

In one embodiment, Ar¹ is an aryl group having an amino-substitutedphenyl core, and has the formula:

wherein r is an integer from 0 to 4, and each R^(A) is independently anaryl substituent, as defined above.

In one embodiment, r is 0. In one embodiment, r is 1.

In one embodiment, r is 2. In one embodiment, r is 3.

In one embodiment, r is 4.

In one embodiment, r is 1 and Ar¹ is a group of the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, Ar¹ is an aryl group having a hydroxy-substitutedphenyl core, and has the formula:

wherein s is an integer from 0 to 4, and each R^(A) is independently anaryl substituent, as defined above, and R^(C), is present, is a reactiveconjugating substituent, or R^(C) is, or contains, a detectable label,as defined above.

In one embodiment, s is 0. In one embodiment, s is 1.

In one embodiment, s is 2. In one embodiment, s is 3.

In one embodiment, s is 4.

In one embodiment, Ar¹ is a group of the formula:

In one embodiment, Ar¹ is a group of the formula:

In one embodiment, Ar¹ is a group of the formula:

In one embodiment, Ar¹ is an aryl group having a naphthyl core, and hasthe formula:

wherein t is an integer from 0 to 3, u is an integer from 0 to 4, andeach R^(A) is independently an aryl substituent, as defined above, andthe compound has the formula:

In one embodiment, Ar¹ is an aryl group having a hydroxy-substitutednaphthyl core, and has the formula:

wherein v is an integer from 0 to 2, u is an integer from 0 to 4, andeach R^(A) is independently an aryl substituent.

In one embodiment, Ar¹ has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the ligand is a compound as described below under theheading “Preferred sulphonated-benzothiazole-like ligands.”

Compounds of the type described above, e.g. of formula (I), for use inthe diagnostic methods of the present invention, may be prepared myconventional means—see e.g. Reference 31.

All such compounds described herein (or derivatives thereof) having theappropriate formula, size, planarity, and activity may be referred togenerally, but not limitatively, hereinafter as ‘sulphonatedbenzothiazole-like compounds’ or ‘SB ligands’). Such compounds willgenerally be ligands of aggregated tau molecules, e.g. those found inpaired helical filaments or neurofibrillary tangles.

The ligands described herein can suitably be detected by incorporating apositron-emitting carbon into one of the methyl groups of the compoundas disclosed herein, and detecting the compound by use of positronemission tomography (PET) as is known in the art. Alternatively, or inaddition, a technetium-containing chelate can be incorporated into thecompound (e.g. as in the R^(c) group of the compounds described herein),so that selective detection of extracellular tangles could be achieved.A preferred chelating group is R^(C)=diethylenetriaminepentaacetic acid.

The ligands may be conjugated, chelated, or otherwise associated, withother chemical groups, dyes, fluorescent markers, antigenic groups,therapeutic moieties, or any other entity which may aid in a prognostic,diagnostic or therapeutic application. For instance, where the ligand isattached to a dye or fluorescent group, the conjugate can be used as alabel of aggregated tau or tau-like molecules. It can thus be used tolabel intracellular or extracellular tangles characteristic of AD.

Phenothiazines

The present inventors have previously identified another class ofcompounds, members of which disrupt the structure of PHFs, and reversethe proteolytic stability of the PHF core (WO 96/30766).

Diaminophenothiazine compounds described in WO 96/30766 are shown by thestructures of FIG. 8 a. Formula (IV) in FIG. 8 a represents differentresonance form of (II) included for clarity. Compounds (II)-(IV) are alloxidised forms while (I) is a reduced form. Such compounds (which may bereferred to hereinafter as ‘diaminophenothiazines’ or ‘phenothiazines’)include, e.g. tolonium chloride and methylene blue. Examples are shownin FIG. 8 b. All of these are shown in the oxidised form, with allexcept thionine being in the form stabilised salts (thionine is shown asa neutral oxidised form).

Compounds which may be used in the methods described herein may be anyhaving a formula shown in FIG. 8 a, wherein:

-   -   each of R₁, R₃, R₄, R₆, R₇ and R₉ is independently hydrogen,        halogen, hydroxy, carboxy, substituted or unsubstituted alkyl,        haloalkyl or alkoxy;    -   R₅ is hydrogen, hydroxy, carboxy, substituted or unsubstituted        alkyl, haloalkyl or alkoxy; and,    -   R₁₀ and R₁₁ are independently hydrogen, hydroxy, carboxy,        substituted or unsubstituted alkyl, haloalkyl or alkoxy;        and pharmaceutically acceptable salts thereof.

In one embodiment:

-   -   each of R₁, R₃, R₄, R₆, R₇ and R₉ is independently hydrogen,        halogen, hydroxy, carboxy, substituted or unsubstituted        C₁₋₆alkyl, C₄₋₄haloalkyl, or C₄₋₆alkoxy;    -   R₅ is independently hydrogen, hydroxy, carboxy, substituted or        unsubstituted C₄₋₆alkyl, C₄₋₄haloalkyl, or C₄₋₆alkoxy;    -   R₁₀ and R₁₁ are independently selected from hydrogen, hydroxy,        carboxy, substituted or unsubstituted C₄₋₆alkyl, C₄₋₄haloalkyl,        or C₁₋₆alkoxy.

The term “alkyl” as used in this respect refers to straight or branchedchain groups, preferably having one to eight, more preferably one tosix, carbon atoms. For example, “alkyl” may refer to methyl, ethyl,n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, tert-pentyl, hexyl, isohexyl, and the like. Suitablesubstituents for the substituted alkyl groups used in the inventioninclude the mercapto, thioether, nitro, amino, aryloxy, halogen,hydroxyl, and carbonyl groups as well as aryl, cycloalkyl and non-arylheterocyclic groups.

The terms “alkoxy” refers to groups as defined herein above as alkylgroups, as the case may be, which also carry an oxygen atom interposedbetween them and the substrate residue to which they are attached.

The term “haloalkyl” represents a straight or branched alkyl chainhaving from one to four carbon atoms with 1, 2 or 3 halogen atomsattached to it. Typical haloalkyl groups include chloromethyl,2-bromethyl, 1-chloroisopropyl, 3-fluoropropyl, 2,3-dibrombutyl,3-chloroisobutyl, iodo-t-butyl, trifluoromethyl and the like.

The “halogen” represents fluoro, chloro, bromo or iodo.

Some of these phenothiazines possess one or more asymmetricallysubstituted carbon atoms and therefore exist in racemic and opticallyactive forms. The invention is intended to encompass the racemic formsof the compounds as well as any of the optically active forms thereof.

Acid addition salts may be formed between basic compounds of FIG. 8 a or8 b and inorganic acids, e.g. hydrohalic acids such as hydrochloric acidand hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid etc.,or organic acid, e.g. acetic acid, citric acid, maleic acid, fumaricacid, tartaric acid, methanesulphonic acid, p-toluenesulphonic acid etc.

In a particular preferred embodiment the present invention employs aphenothiazine wherein:

R₁, R₃, R₄, R₆, R₇ and R₉ are independently —H, —CH₃, —C₂H₅, or —C₃H₇;R₁₀ and R₁₁ are independently —H, —CH₃, —C₂H₅ or —C₃H₇;

R₅ is —H, —CH₃, —C₂H₅, or —C₃H₇;

or a pharmaceutically acceptable salt thereof.

The present inventors now teach herein that such phenothiazine compoundsof this sort can bind to the PHFs at a specific site which appears, onthe basis of its binding characteristics, to be distinct from that atwhich the sulphonated benzothiazole-like compounds, described above, canbind. The binding of the phenothiazine compounds to this site is thoughtto effect the inhibition of tau aggregation.

Phenothiazine compounds may be used in the methods and other embodimentsdescribed above, incorporating labels as appropriate. When suitablylabeled with a positron-emitting functional group (detectable by PET—seeFIGS. 11, 11 b, 12, 13) such compounds would serve as ligands for alltau aggregates, and would be capable of crossing the blood-brain-barrier(Ref 36) and entering cells.

In a further embodiment, in the light of the disclosure herein, it willbe appreciated that the effect of, and particularly the progress of,therapy based on tau-tau binding inhibitors may be monitorable by use ofSB ligands.

Blocking Ligands

Preferably these are benzthiazoles of the formula:

wherein:n is an integer from 0 to 4;each R^(BT) is independently a blocking ligand benzothiazolesubstituent;m is an integer from 0 to 4;each R^(P) is independently a phenylene substituent;each R is independently —H or an amino substituent; and, either:R^(N) and X⁻ are both absent and the associated (tertiary) nitrogen atomis neutral; or:R^(N) is a benzothiazolino substituent and the associated (quaternary)nitrogen atom bears a positive charge, and X⁻ is a counter ion.

Preferred benzthiazoles include thioflavin T. As shown in the Examplesbelow, such compounds (e.g. 1b or 2 in FIG. 5) are displaced from NFTsby SB-ligands (e.g. 1a in FIG. 5). However such compounds do bindpreferentially to amyloid.

In one embodiment, n is 0. In one embodiment, n is 1.

In one embodiment, n is 2. In one embodiment, n is 3.

In one embodiment, n is 4. In one embodiment, n is 0, 1, or 2.

Examples of blocking ligand benzothiazole substituents, R^(BT), include,but are not limited to, C₁₋₄alkyl groups, —SO₃H, and —SO₃M³, wherein M³is a cation. In one embodiment, M³ is an alkali metal cation. In oneembodiment, M³ is Li, Na, K, or Cs. In one embodiment, M³ is Na or K.Examples of C₁₋₄alkyl groups include, but are not limited to, -Me, -Et,-nPr, and -iPr.

In one embodiment, each R^(BT) is independently a C₁₋₄alkyl group.

In one embodiment, each R^(BT) is selected from: -Me, -Et, -nPr, and-iPr. In one embodiment, each R^(BT) is -Me. In one embodiment, n is 1and R^(BT) is -Me, -Et, -nPr, or -iPr. In one embodiment, n is 1 andR^(BT) is Me.

In one embodiment, one of the R^(BT) groups is —SO₃H or —SO₃M³. In oneembodiment, one of the R^(BT) groups is —SO₃H or —SO₃M³, and another ofthe R^(BT) groups is a C₁₋₄alkyl group. In one embodiment, n is 2 andone R^(BT) is a C₁₋₄alkyl group and one R^(BT) is —SO₃H or —SO₃M³. Inone embodiment, n is 2 and one R^(BT) is -Me and one R^(BT) is —SO₃H or—SO₃M³.

In one embodiment, R^(N) and X⁻ are both absent and the associated(tertiary) nitrogen atom is neutral.

In one embodiment, R^(N) is a benzothiazolino substituent and theassociated (quaternary) nitrogen atom bears a positive charge, and X⁻ isa counter ion. Examples of benzothiazolino substituents, R^(N), include,but are not limited to, C₁₋₄alkyl groups. In one embodiment, R^(N) is-Me, -Et, -nPr, or -iPr. In one embodiment, R^(N) is -Me. Examples ofcounter ions include, but are not limited to, Cl⁻, Br⁻, and I⁻. In oneembodiment, R^(N) is -Me and X⁻ is Cl⁻.

In one embodiment, m is 0. In one embodiment, m is 1.

In one embodiment, m is 2. In one embodiment, m is 3.

In one embodiment, m is 4.

Examples of phenylene substituents, R^(P), include, but are not limitedto, C₁₋₄alkyl groups.

In one embodiment, each R is —H, and the amino group is —NH₂. In oneembodiment, one R is —H and one R is an amino substituent. In oneembodiment, each R is an amino substituent. Examples of aminosubstituents include, but are not limited to, C₁₋₄alkyl groups. In oneembodiment, the amino group is —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr),—NMe₂, —NEt₂, N(iPr)₂, or —N(nPr)₂.

Preferred embodiments of blocking ligands are shown in FIG. 5 ascompounds 1b and 2.

Preferred Sulphonated-Benzothiazole-Like Ligands

In one aspect of the present invention, the ligands used to label theaggregated tau, preferable extracellular aggregated tau present in NFTs,are compounds having the formula (II):

wherein:M¹ is an alkali metal cation;n is an integer from 0 to 3;each R^(BT) is a independently benzothiazole substituent;m is an integer from 0 to 4;each R^(RL) is independently a rigid linker aryl substituent;s is an integer from 0 to 4;each R^(A) is independently an aryl substituent; and,R^(C), if present, is a reactive conjugating substituent, orR^(C) is, or contains, a detectable label.

In various embodiments, M¹, n, each R^(BT), each R^(RL), s, each R^(A),and R^(C) are as described herein (e.g., under the heading“Sulphonated-benzothiazole-like ligands,” above).

The rigid linker group, RL, and the aryl group, Ar¹, together with thebenzothiazole group, form a compound which is substantially planar, thatis, has a high degree of planarity.

As shown herein, such compounds may be particularly effective when it isdesired to incorporate a bulky R^(C) group in order to facilitatedetection.

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In various embodiments, s may be as discussed above.

In one embodiment, each R^(A) is independently selected from thesubstituents given above in relation to formula (I).

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

Various R^(C) substituents are discussed elsewhere herein.

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

In one embodiment, the compound has the formula:

Some of these preferred compounds are shown in FIGS. 4 a-c, andderivatives thereof.

Thus, according to one aspect, the present invention provides a compoundrepresented by the formula shown in FIG. 4 a, or a derivative thereofe.g. wherein R^(c) is a conjugating group as described above. As shownin the Examples hereinafter, such derivatives (e.g. compound 4b) retainthe appropriate binding activity.

The novel compounds disclosed (e.g. of formula (II)) herein are usefulinter alia as synthetic ligands of neurofibrillary tangles, such asthose characteristic of AD. The discovery of the minimum criticalstructure required for binding to these tangles thus provides for thepossibility of designing high-affinity ligands which can be used totarget the tangles, and can thus be used in the diagnosis, prognosis ortreatment of diseases such as AD.

Such compounds will be referred to below as preferred SB ligands.

Mimetics of Preferred SB Ligands

In general, there are several steps commonly taken in the design of amimetic from a compound having a given target property (in this case apreferred SB tau-tau aggregation ligand) of which the most important isthat the particular parts of the compound that are critical and/orimportant in determining the target property are determined. Theprovision by the present inventors of the minimum critical structurerequired for high affinity binding to aggregated tau molecules hasobviated this step.

The minimum critical structure of compound 4a can be modeled accordingto its physical properties, e.g. stereochemistry, bonding, size and/orcharge, using data from a range of sources, e.g. spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of the ligand,rather than the bonding between atoms) and other techniques can be usedin this modeling process.

In a variant of this approach, the three-dimensional structure of thepreferred SB ligand and its binding partner are modeled. This can beespecially useful where the ligand and/or binding partner changeconformation on binding, allowing the model to take account of this inthe design of the mimetic. A template molecule is then selected ontowhich chemical groups which mimic the minimum critical structure can begrafted. The template molecule and the chemical groups grafted on to itcan conveniently be selected so that the mimetic is easy to synthesise,is likely to be pharmacologically acceptable, and does not degrade invivo, while retaining the required biological activity. The mimetic ormimetics found by this approach can then be screened to see whether theyhave the target property, or to what extent they exhibit it. Furtheroptimisation or modification can then be carried out to arrive at one ormore final mimetics for further testing or optimisation, e.g. in vivo orclinical testing. Optimisation may include selecting a mimetic compoundas described above, and contacting it with a preparation of aggregatedtau molecules (e.g. preaggregated tau in solution, or bound to a solidphase, or isolated from PHFs—see WO96/30766 and assays described below)and determining the extent to which the test substance(s) binds to theaggregated tau molecules and\or displaces compound 4a from themolecules.

Methods of Labelling Aggregated Tau

In one aspect, the present invention thus provides a method of labellingaggregated tau or tau-like molecules, comprising contacting theaggregated tau molecules with a preferred SB-ligand compound orderivative thereof as provided herein (e.g. of formula (II)) anddetecting the presence of said compound or derivative. Methods of usemay be performed e.g. by analogy to the use of the ligands given in Refs26-34.

Where used herein, the term “tau protein” refers generally to anyprotein of the tau protein family. Tau proteins are characterised asbeing one among a larger number of protein families which co-purify withmicrotubules during repeated cycles of assembly and disassembly(Shelanski et al. (1973) Proc. Natl. Acad. Sci. USA, 70., 765-768), andare known as microtubule-associated-proteins (MAPs). Members of the taufamily share the common features of having a characteristic N-terminalsegment, sequences of approximately 50 amino acids inserted in theN-terminal segment, which are developmentally regulated in the brain, acharacteristic tandem repeat region consisting of 3 or 4 tandem repeatsof 31-32 amino acids, and a C-terminal tail.

“Tau like” molecules include, for instance, MAP2, which is thepredominant microtubule-associated protein in the somatodendriticcompartment (Matus, A., in “Microtubules” [Hyams and Lloyd, eds.] pp155-166, John Wiley and Sons, NY). MAP2 isoforms are almost identical totau protein in the tandem repeat region, but differ substantially bothin the sequence and extent of the N-terminal domain (Kindler and Garner(1994) Mol. Brain. Res. 26, 218-224). Nevertheless, aggregation in thetandem-repeat region is not selective for the tau repeat domain. Thus itwill be appreciated that any discussion herein in relation to tauprotein or tau-tau aggregation should be taken as relating also totau-MAP2 aggregation, MAP2-MAP2 aggregation and so on.

The preferred SB ligand may be conjugated, chelated, or otherwiseassociated with, a further group or entity which has a diagnostic,prognostic or therapeutic purpose or effect, e.g. to a fluorescent groupwhich thus enables visualisation of neurofibrillary tangles to which theligand binds.

Diagnostic Compositions and Uses

Generally, a preferred SB ligand according to the present invention(e.g. of formula (II)) may be provided in an isolated and/or purifiedform, i.e. substantially pure. This may include being in a compositionwhere it represents at least about 90% active ingredient, morepreferably at least about 95%, more preferably at least about 98%. Sucha composition may, however, include inert carrier materials or otherpharmaceutically- and physiologically-acceptable excipients. Acomposition according to the present invention may include in additionto a preferred SB ligand as disclosed herein, one or more othermolecules of diagnostic, prognostic or therapeutic use.

A preferred SB ligand substance according to the present invention, or acomposition comprising such a ligand, may be provided for use in amethod of diagnosis, prognosis or treatment of the human or animal bodyby therapy, especially in relation to a condition such as AD asdescribed below.

In a further aspect, the present invention provides a method ofdiagnosis or prognosis, the method comprising administering to themammal a diagnostically- or prognostically-effective amount of one ormore preferred SB ligands as described herein. This aspect embraces suchcompounds for use in a method of diagnosis or prognosis. Both in vitroand in vivo uses are encompassed by this aspect. In vitro methods may beperformed by (i) obtaining a sample of appropriate tissue from asubject; (ii) contacting the sample with the preferred SB ligand; (iii)detecting the amount and\or localisation of the preferred SB ligandbound to the sample (iv) correlating the result of (v) with the stage orseverity of the disease in the subject.

In a further aspect, the present invention provides the use of apreferred SB ligand or derivative as provided herein, in the manufactureof a composition for the diagnosis, prognosis or therapy of a disease asdescribed above.

The disease or condition may be e.g. AD, or an AD-like condition, or anyother condition in which aggregated protein molecules are implicated.

Notably it is not only Alzheimer's Disease in which tau protein (andaberrant function or processing thereof) may play a role. Thepathogenesis of neurodegenerative disorders such as Pick's disease andProgressive Supranuclear Palsy (PSP) appears to correlate with anaccumulation of pathological truncated tau aggregates in the dentategyrus and stellate pyramidal cells of the neocortex, respectively. Otherdementias include fronto-temporal dementia (FTD); parkinsonism linked tochromosome 17 (FTDP-17); disinhibition-dementia-parkinsonism-amyotrophycomplex (DDPAC); pallido-ponto-nigral degeneration (PPND); Guam-ALSsyndrome; pallido-nigro-luysian degeneration (PNLD); cortico-basaldegeneration (CBD) and others (see Wischik et al. 2000, loc. cit, fordetailed discussion—especially Table 5.1). All of these diseases, whichare characterized primarily or partially by abnormal tau aggregation,are referred to herein as “tauopathies”.

Diagnostic compositions may comprise, in addition to one of the aboveSB-ligand derivatives, a diagnosticly acceptable excipient, carrier,buffer, stabiliser, or other materials well known to those skilled inthe art. Such materials should be non-toxic and should not interferewith the binding activity of the substance to aggregated tau, or theefficacy of any bioactive group linked to or otherwise associated withthe substance. The precise nature of the carrier or other material maydepend on the route of administration, e.g. oral, intravenous, cutaneousor subcutaneous, nasal, intramuscular, intraperitoneal routes.

Diagnostic compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may include a solid carriersuch as gelatin or an adjuvant. Liquid diagnostic compositions generallyinclude a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,dextrose or other saccharide solution or glycols such as ethyleneglycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the ligand will be in the form of a parenterallyacceptable aqueous solution which is pyrogen-free and has suitable pH,isotonicity and stability. Those of relevant skill in the art are wellable to prepare suitable solutions using, for example, isotonic vehiclessuch as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

A composition as described above may be administered alone or incombination with other treatments, either simultaneously orsequentially, depending on the condition to be treated.

Identification of Ligands

A further method for identifying ligands for aggregated tau requires ascreening assay which can be used in a format which permits highthrough-put screening of chemical libraries to identify compounds withthe required activity. Until now, no such method has been readilyavailable. Preferred methods would not require pre-labelled compounds,since the labelling process may severely limit chemical search capacity.

In a further aspect of the present invention a method is provided whichcan be used as a high through-put screening assay, which is not limitedby the need to have pre-labelled test substances. In preferredembodiments the method employs the following steps:

1. the high capacity generation of tau proteins in a form which haveundergone partial aggregation in the course of preparation;2. using tau proteins prepared in this manner for testing putativeligands in the tau-tau binding assay provided in WO96/30766 to identifysubstances which have minimal or entirely absent activity astau-aggregation inhibitors or enhance tau-tau binding at highconcentrations;3. testing putative ligands in the presence of an exemplary potenttau-aggregation inhibitor, such as DMMB, at an inhibitory concentration;4. putative ligands can be identified by the property that they lackcapacity to block tau-tau binding through the repeat domain, but blockthe inhibitory activity of a potent tau-aggregation inhibitor.

Thus the invention provides an in vitro method for identifying a ligandcapable of labeling aggregated PHF tau protein, the method comprisingthe steps of:

(i) providing a first agent suspected of being capable of labelingaggregated PHF tau protein,(ii) contacting (a) a tau protein or a derivative thereof containing thetau core fragment bound to a solid phase so as to expose a high affinitytau capture site (e.g. a truncated tau protein corresponding to the corefragment and terminating at Ala390-dGA), with (b) a liquid phase tauprotein or derivative thereof capable of binding to the solid phase tauprotein or derivative (e.g. dGAE which terminates at Glu-391), and (c)said selected first agent and (d) a second agent known to be tau-taubinding inhibitor,(iii) selecting a first agent which fully or partially relieves theinhibition of binding of the liquid phase tau protein or derivative of(b) to the solid phase tau protein or derivative of (a) by the inhibitor(d).

Agents satisfying (iii) may be provided as ligands.

Preferably the method is carried out in conjunction with (before,during, after) the following steps:

(ibis) contacting (a) a tau protein or a derivative thereof containingthe tau core fragment bound to a solid phase so as to expose a highaffinity tau capture site, (b) a liquid phase tau protein or derivativethereof capable of binding to the solid phase tau protein or derivative,with (c) said first agent and,(ibis.1) detecting inhibition of tau-tau binding as exhibited byinhibition of binding of the liquid phase tau protein or derivative of(b) to the solid phase tau protein or derivative of (a),(ibis.2) selecting a first agent which has minimal or absent activity astau-tau binding inhibitors and\or optionally enhance tau-tau binding.

Agents satisfying (iii) and (ibis.2) may be provided as ligands.

The inhibitor is preferably a diaminophenathiozine as described above(most preferably DMMB). The compounds selected for screening may be anycompound, including SB-ligands.

In preferred forms the liquid phase tau protein or derivative isprepared in a form which has undergone partial aggregation prior toexposure to the solid phase. Apart from that, the assay may be carriedout broadly as described in WO96/30766 and summarised in more detail inthe Examples below. Preferably alkaline or physiological conditions(e.g. PBS) are used for the binding steps, and results are detectedimmunologically.

These and other aspects of the present invention will become moreapparent on reading the ensuing non-limiting Examples, in whichembodiments of the invention will be described by way of example only.Reference is made to the accompanying figures, in which:

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the regional distribution of PHF-tau, measured usingantibodies mAb423 (A) or mAb7.51 after formic acid treatment of the PHFfraction (B), for 18 cases of AD. Taken from Mukaetova-Ladinska et al.,(1993), Am. J. Pathol. 143, 565-578.

FIG. 2 (a) shows the aggregation of tau molecules and the appearance ofneurofibrillary tangles during the pathological stages of AD. Taken from(Mukaetova-Ladinska, E. B. et al. (2000) Am. J. Pathol. Vol. 157, No. 2,623-636); (b) shows the neuropathological staging proposed by Braak; (c)shows that the appearance of clinical dementia by DSM-IV criteriaappears to correspond statistically to the transition between stages IIIand IV;

(d) shows levels of SDS-insoluble beta-amyloid protein isolated fromcontrol cases and cases with Alzheimer's disease, as reported inHarrington et al., (Am J Pathol 1994; 145: 1472-1484). Although the meanlevel is higher in AD than in controls, 70% of AD cases overlap withlevels of beta-amyloid found in control subjects.

FIG. 3 shows a schematic representation of a neurofibrillary tangle(top), and the immunoreactivity changes that are observed during diseaseprogression (bottom). Taken from Bondareff et al. (1994) J. Neuropathol.Exp. Neurol. 53, 2, 158-164.

FIG. 4 shows the chemical structures of: the minimum critical structurewhich enables high affinity binding to aggregated tau molecules(compound 4a); a biotinylated version of compound 4a (compound 4b); andan R-substituted derivative of compound 4a (compound 4c), wherein R isany suitable substituent.

FIG. 5 shows the chemical structures of: primulin (compound 1a);thioflavin-T (compound 1b); 2-(4-amino phenyl)-6-methyl-1-sulphonatebenzothiazole (compound 2); thiazin red (compound 3a); and thiazinyellow (compound 3b).

FIG. 6 shows the fluorescence peaks of primulin in solution (left), andwhen bound within a pure preparation of PHFs (right).

FIG. 7 shows the fluorescence peaks of primulin bound to PHFs, in theabsence (left) and presence (right) of citraconic anhydride. As isshown, citraconic anhydride has the effect of disrupting the orderlystructure of PHFs and releasing free tau and free unbound primulin.Citraconic anhydride also has the effect of reversing the charge onlysine residues and this may also play a role in releasing the primulin.

FIGS. 8 a and 8 b illustrate exemplary tau-tau binding inhibitors, asdisclosed in WO 96/30766.

FIG. 9 shows the fluorescence of primulin bound to PHFs in the presenceof alcian blue. The figure demonstrates that, in the presence of alcianblue, a disrupter of PHF structure (Ref 33), there is no disturbance ofthe characteristic bound primulin fluorescence peak at 460 nm.

FIG. 10 shows the effect of various compounds (“MR”, molar ratio ofcompound:tau) on tau-tau binding using tau proteins prepared accordingthe method provided in WO96/30766 and referred to herein as “Preparation1”.

FIG. 11 a show schematically the synthesis of [¹¹C]-labelled methyleneblue. The reaction proceeds via N-methylation of the amines with[¹¹C]iodomethane. HPLC can be used to purify the desired product fromany by-products. FIG. 11 b shows a further synthesis based on thionin,NaH and CH₃I.

FIG. 12 shows schematically the synthesis of [¹¹C]-labelled Azure B. Thereaction proceeds via N-methylation of the amines with [¹¹C]iodomethane.HPLC can be used to purify the desired product from any by-products.

FIG. 13 shows schematically the synthesis of a [¹⁸F]-labelled derivativeof compound 4a of FIG. 4. The reaction proceeds via nucleophilicaromatic substitution whereby a nitro-group on a precursor compound isexchanged for a [¹⁸F] fluoro group. HPLC can be used to purify thedesired product from any by-products.

FIG. 14 shows the structures Primulin, Benthothiazole Analogue andThiazin Yellow. The sizes of these molecules have been determined on thebasis of C—C distances known from chrystal structures, and designated Aand B for each molecule. The C—C lengths are as follows:

Primulin Minimum 14.78 AU Maximum 15.11 AU Mean 14.95 AU AnalogueMinimum 15.05 AU Maximum 15.26 AU Mean 15.17 AU Thiaz. Yellow Minimum15.73 AU Maximum 16.14 AU Mean 15.93 AU

FIGS. 15 and 16 illustrate the crystal structure of the ‘B’ part of theprimulin structure (Soon-Beng Teo et al., 1995, Acta Crystallogr., Sect.C, 591.

FIGS. 17 and 18 illustrate the crystal structure of a compound denotedN2A (Gilardi, R. D., 1972, Acta Chrystallogr., Sect. B, 107).

FIGS. 19 and 20 show the crystal structure of a diazoaminobenzene(Gladkova & Kondrashev, 1972, Kristallografiya (41) 17 33.

FIGS. 21 and 22 illustrate how the molecule of FIGS. 15 and 16crystalises in space.

FIG. 23 shows a comparison of the mean, maximum and minimum extents ofmolecules which are active ligands (primulin and “analog”), and thiazinyellow (which is inactive as a ligand). The dimensions are given inangstrom units (AU).

FIG. 24 shows a similar comparison for the basic benzothiazole nucleus(i.e. molecules 1b and 2 of FIG. 5) and the diaminophenothiazines. Thesedistances are carbon-to-carbon distances.

FIG. 25 shows probability of extracellular tangles as a function ofBraak staging. Stages 2-4 can be clearly distinguished from stage 1 onthe basis of probability of extracellular tangles in E2/Trans and E4/HC.

FIG. 26 shows probability of intracellular tangles as a function ofBraak staging. Intracellular tangles provide a poor basis fordiscrimination of early stages in these regions, but a good basis fordiscriminating stages 4 and 5 using neocortical regions.

FIG. 27 corresponds to FIG. 25, but wherein cases with MMSE scoresgreater than 21 in the 12 months prior to death were selected. Similarresults are obtained.

FIG. 28 corresponds to FIG. 26, but using cases with MMSE scores greaterthan 21 in the 12 months prior to death were selected. Similar resultsare obtained.

FIG. 29 shows extracellular tangle densities (counts per mm²) as afunction of Braak staging. Similar results are obtained to those shownin FIG. 25.

FIG. 30 shows intracellular tangle densities (counts per mm²) as afunction of Braak staging.

FIG. 31 a shows a barely visible tangle visualised with thioflavin-T at0.001% (arrowhead). In ifI suspensions such as this, tangles can be seenby blue fluorescence which is not distinct from that associated withbinding of contaminants in the preparation. The bottom panel shows thatblue tangle fluorescence produced by thioflavin-T at 0.001% is displacedby yellow tangle fluorescence produced by primulin at 0.001%. FIG. 31 bshows Electron-microscopy of PHFs labelled after Pronase digestion. (a)Chemical labelling with the biotinylated benzothiazole analogue shown as4b in FIG. 4. PHFs were deposited on a carbon-coated grid after Pronasedigestion, and incubated briefly with compound 4b, and then incubatedwith an anti-biotin antibody preparation that had been conjugated withcolloidal gold. Decoration of isolated PHFs establishes that thecompound 4b binds to the proteolytically stable PHF structure. (b) mAb423 immunolabelling of isolated PHFs after Pronase digestion., followedby incubation with a gold-conjugated goat anti-mouse second antibody asdescribed in Novak et al. 1993 (Novak M, Kabat J, Wischik CM (1993)“Molecular characterisation of the minimal protease-resistant tau unitof the Alzheimers' disease paired helical filament”, EMBO J. 12:365-370). As shown in this paper, mAb 423 labels PHFs derived fromintracellular tangles (which preserve N-terminal tau immunoreactivity inthe fuzzy outer coat) poorly, but Pronase digested PHFs strongly.Likewise Mena et al. (1996) (Mena R, Edwards P C, Harrington C R,Mukaetova-Ladinska E B, Wischik C M, “Staging the pathological assemblyof truncated tau protein into paired helical filaments in Alzheimer'sdisease”, Acta Neuropathol 91: 633-641) shows that in intracellulartangles mAb 423 immunoreactivity is largely occluded, but can be exposedby formic acid pretreatment of sections.

FIG. 32 shows “Preparation 2” of purified tau protein as described inExample 7 below.

FIG. 33 shows a graphical plot of the results of a preparative run fordGA. “Purification-fold” is expressed as the ratio of specificimmunoreactivity for each fraction (ie immunoreactivity/proteinconcentration) to specific immunoreactivity in the DE flow-through.

FIG. 34 shows gel filtration chromatography of purified dGAE. Apparentelution size in non-denaturing conditions:

-   1—˜320 kD; 2—˜80 kD; 3—˜30 kD; 4—˜10 kD    About 64% of mAb 7.51 immunoreactivity elutes in fractions    corresponding to species of size>15 kD.

FIG. 35 shows gel filtration chromatography of purified T40. Apparentelution size in non-denaturing conditions:

-   1—˜450 kD; 2—˜160 kD; 3—˜55 kD.

About 50% of mAb 499 immunoreactivity elutes in fractions correspondingto species of size>60 kD.

FIG. 36 shows thionine activity against tau-tau binding in Preparations1 & 2.

FIG. 37 shows tolonium chloride activity against tau-tau binding inPreparations 1 & 2.

FIG. 38 shows DMMB activity against tau-tau binding in Preparations 1 &2.

FIGS. 39 a-c show that full-length tau protein (hT40) prepared accordingto the Preparation 2 protocol demonstrated minimal tau-tau bindingactivity when used in the aqueous-phase with dGA in the solid-phase (b).However, when hT40 was used in the solid-phase (c), binding of dGAE wassimilar to that obtained for binding of dGAE to dGA in the solid-phase(a).

FIG. 40 shows primulin and thiazin red have no inhibitory activityagainst tau-tau binding in Preparation 2, and indeed enhance suchbinding at high concentrations.

FIG. 41 shows blocking of inhibitory effects of 5 μM DMMB on tau-taubinding in the presence of increasing concentrations of Primulin (a) andThiazin Red (b), expressed as “molar excess” relative to DMMB. Similarresults are shown for 15 μM DMMB in the presence of increasingconcentrations of Primulin (c) and Thiazin Red (d).

FIG. 42 shows attenuation and reversal of inhibition of Tau-Tau bindingby DMMB in the presence of increasing molar excess of Primulin. For eachgraph, Tau-Tau binding is shown in the presence of constantconcentrations of DMMB co-incubated with Primulin at 0×, 1×, 5×, 10×,100× the DMMB concentrations shown. Inhibition of Tau-Tau bindingproduced by DMMB is progressively attenuated and reversed in thepresence of increasing molar excess of Primulin.

FIG. 43 shows a tau-Tau binding curve in the presence of 25 μM DMMB andincreasing Primulin molar excess as shown. Tau-Tau binding can bemodelled as follows:

Binding=(BMax×[Prim])/(Kd+[Prim])

-   -   Where        -   B_(Max)=1.67        -   Kd=13.37        -   r=0.977 (observed vs predicted)

FIG. 44 shows a tau-Tau binding curve in the presence of 5 μM DMMB andincreasing Primulin molar excess as shown. Tau-Tau binding can bemodelled as follows:

Binding=(BMax×[Prim])/(Kd+[Prim])

-   -   Where        -   B_(Max)=1.38        -   Kd=13.86        -   r=0.927 (observed vs predicted)

FIG. 45 shows a tau-Tau binding curve in the presence of 5 μM DMMB andincreasing Thiazin Red molar excess as shown. Tau-Tau binding can bemodelled as follows:

Binding=(BMax×[TR])/(Kd+[TR])

-   -   Where        -   B_(Max)=1.64        -   Kd=17.45        -   r=0.915 (observed vs predicted)

EXAMPLES Methods and materials PHF-Binding Compounds

Compounds used herein were supplied by ICI Pharmaceuticals unless statedotherwise. Thioflavin-T and thiazine yellow were purchased from FlukaAG.

Quantitation of Fluorescence

Serial 16 μm sections are cut from the hippocampus of a case dying withclinically and neuropathologically confirmed AD. These sections werestained with thioflavin-S at concentrations 0.01%, 0.001% or 0.0001% inwater for 5-10 min, then washed in water, and mounted in Apathe'saqueous medium. In a second series of experiments, sections were cutfrom the hippocampus and nucleus basalis of Meynert. These sections werestained with primulin at concentrations 0.1%, 0.01%, 0.001% and 0.00001%in water for 5-10 min, then washed in water, and mounted in Apathe'saqueous medium.

A Leitz fluorescence microscope fitted with a photo-multiplyer tube(Model MPV-2) was used to quantitate fluorescence emission. Three Leitzfilter blocks were used as follows:

1. Filter block H2, code 513 417

-   -   Excitation range Band pass 390-490 nm    -   Mirror RKP 510 (i.e. transmit below 510 nm)    -   Suppression filter LP 515 (i.e. reflect above 515 nm)        3. Filter block G, code 513 416    -   Excitation range Band pass 350-460 nm    -   Mirror RKP 510 (i.e. transmit below 510 nm)    -   Suppression filter LP 515 (i.e. reflect above 515 nm)        4. Filter block A, code 513 410    -   Excitation range UV band pass 340-380 nm    -   Mirror RKP 400 (i.e. transmit below 400 nm)    -   Suppression filter LP 430 (i.e. reflect above 430 nm)

Preparation of if I and II

IfI material was prepared as described by Wischik et al (1985) J CellBiol 100: 1905-1912.

IfII material was prepared as described by Wischik et al (1995)Neurobiol Aging 16: 409-431. For experiments involving non-pronasedigested ifII an identical protocol was followed, omitting the pronasedigestion step.

Spectrofluorometry of ifII

These measurements were carried out in a Perkin-Elmer spectrofluorimeter(model MPF-3). A concentration of ligand of 0.00001% was routinely usedfor all measurements. Primulin was found to have an excitation peak at370 nm and an emission peak at 515 nm. All measurements were thereforecarried out at a standard excitation wavelength of 370 nm, and aconstant slit width of 3 mm.

Competitive Binding Assay

IfI material was homogenised in a 0.2 ml glass homogeniser in PBS. Tothe suspension, test compounds were added to final concentrationsranging from 0.1% to 0.00001%. These were allowed to incubate for 5 minand primulin was added at equivalent or lower concentration. Thesuspensions were transferred to a glass slide, and examined byfluorescence microscopy across a range of fluorescence filter blocks,covering excitation and emission wavelengths between 380 nm and 570 nm.The end point sought in these observations was displacement of typicalprimulin fluorescence from tangle fragments.

Ligand Electronmicroscopy

PHFs derived from an ifI fraction were deposited on a carbon coated gridafter pronase digestion, and incubated briefly with a preparation ofbiotinylated Primulin, and then incubated with an anti-biotin antibodythat has been conjugated with colloidal gold by the method of Slot andGueze (1981).

Succinylation and Chromatography of IfII

Washed ifII fractions were taken up in 8 M urea/50 mM borate (1 ml, pH9) and sonicated, 1 ml succinic anhydride in acetone was added to afinal concentration of 250 mM succinate in 4 ml, and the pH wasmaintained at 8.5 with sodium hydroxide. The solution was clarified bycentrifugation and applied to a Sephacryl 5200 column equilibratedbicarbonate. The column eluate was monitored at either 230 or 280 nm.

Because succinylated fractions could not be visualised by Coomassiestaining or silver staining of gels, bands were detected byautoradiography after specific chemical labelling of ifII fractions withBolton-Hunter reagent (Amersham).

For photoaffinity labelling of PHF derived peptides, ifI or ifIIfractions were pre-incubated with an I¹²⁵-labelled photolabilederivative.

The photolabelled fraction running at a Kav of 0.21 was concentrated byultrafiltration through an Amicon YM2 membrane (10 ml), digested withchymotrypsin (0.01 mg/ml) in 50 mM ammonium bicarbonate. Chymotrypticfragments for sequence analysis were isolated by Dr H. C. Thogersen byreverse phase HPLC using a C18 column, with a 0-100% acetyl nitrilegradient, with 0.1% trifluoroacetic acid. Chymotryptic peptides weresequenced.

Morphological Studies of PHFs in the Presence of Phenothiazines

For these experiments, ifII fractions were prepared as described abovefor electron microscopy. This material was either incubated directlywith preparations of phenothiazines at final concentrations rangingbetween 0.1% and 0.0001% and then applied to carbon coated grids, andexamined directly after LiPTA staining (1%). Alternatively, ifIIsuspensions were deposited on carbon coated grids, partially dried, andwashed with solutions of phenothiazine. Such preparations were eitherstained directly with LiPTA or were processed further for immunoelectronmicroscopy using 6.423 as the primary antibody. Electron micrographswere recorded at nominal magnifications between 25,000 and 45,000.

Calculation of aggregated tau protein in the extracellular spaceexpressed in μg/g of brain tissue as a function of Braak stagingPreviously reported PHF-tau levels in pmo1/g (P) and tangle counts permm² (T) in a clinically and neuropathologically staged cohort (R. Y. K.Lai, et al., Neurobiol Aging 16, 433 (1995)) were used to derive anestimate of PHF-tau level per affected pyramidal cell (PC) in pg/cellusing the same ELISA in human brain. The tangle count per mm² providesan estimate of the number of affected pyramidal cells within a volume 1mm×1 mm×0.1 mm (0.0001 cm³), allowing that any tangle profile counted ina nominal 7 μm section could extend ˜45 μm orthogonal to the section ineither direction (S. M. Blinkov, I. I. Glezer, The human brain infigures and tables, a quantitative handbook; Plenum Press, NY, 1968,Table 204). The core PHF-tau level in pg/cm³ is 10×P since the PHF-coretau fragment is 10 kD (C. M. Wischik, et al., Proc. Natl. Acad. Sci. USA85, 4506 (1988)). From this, PC=(P×10)/(T/0.0001). At Braak stages 4-6(H. Braak, E. Braak, Acta Neuropathol. 82, 239 (1991)), regional valuesfor PC in grey matter were: frontal cortex, 0.13±0.05 pg/cell;hippocampus, 0.60±0.39 pg/cell; temporal cortex, 1.074±0.44; entorhinalcortex 1.56±0.63 pg/cell. These differences reflect anatomicaldifferences, different regional rates of disease progression (C.Bancher, H. Braak, P. Fischer, K. Jellinger, Neurosci. Lett. 162, 179(1993), also Gertz et al., Acta Neuropathol. 95, 154 (1988)), and thedegree to which tangle counts underestimate PHFs accumulating indystrophic neuritis at more advanced stages of pathology (Lai et al,1995, loc cit). The overall means provide an approximation for thePHF-levels per cell which would be relevant to AD. These are 0.37±0.08pg/cell for cases at Braak stages 1-3, and 1.08±0.28 pg/cell for casesat Braak stages 4-6.

For the purpose of estimating extracellular aggregated PHF tau, shownbelow in the Table, and in FIGS. 26, 27, 29 and 31, tangles were countedas extracellular if mAb 423 immunoreactivity could be demonstrated aftertreatment of glass-mounted sections with 98% formic acid for 5 minutesprior to incubation with mAb 423 for 1 hr. For the avoidance of doubt,this methodology differs from that reported in Mena et al. (1996) wherefree-floating vibrotome sections were incubated briefly with formicacid, and then overnight with mAb 423. As shown in that report, thislatter overnight free-floating section protocol achieved maximal mAb 423immunoreactivity in intracellular tangles, and showed that allintracellular tangles contain mAb 423 immunoreactivity, albeit in astate substantially occluded by the fuzzy outer coat of the PHF (seeFIG. 3). The purpose of the present protocol was to ensure maximumlabelling of the Stage 3 and Stage 2 tangles illustrated in FIG. 3. Someminor degree of labelling of intracellular tangles could not be entirelyexcluded, and counting attempts have also been made where a subjectivediscrimination has been attempted. However, the latter estimates do notagree with density or probability of labelling of tangles by mAb AT8(FIGS. 28, 30, 32), which is solely intracellular, and shows completelydifferent profiles with respect neuropathological stage from thoserevealed using mAb 423. For the purposes of the present calculations,therefore, counts of mAb 423-immunoreactive tangles were taken assubstantially or entirely representative of extracellular tanglepathology at Stages 2 and 3 as shown in FIG. 3, but substantially notStage 1 of FIG. 3. For the avoidance of doubt, the Stages referred to inFIG. 3 are not Braak stages, but stages of degeneration of a singleneurone containing a tangle.

The specific data shown in Table 1 was based on the following:

Number BST ME1T4 PC PT4 REG3B SE1T4 1 1.0000 0.3982 1.5600 0.6212 1.00000.3982 2 2.0000 6.7259 1.5600 10.4924 1.0000 2.7047 3 3.0000 14.96461.5600 23.3448 1.0000 2.9836 4 4.0000 33.6297 1.5600 52.4624 1.000010.9883 5 5.0000 44.3102 1.5600 69.1240 1.0000 13.0298 6 1.0000 0.00.6000 0.0 2.0000 0.0 7 2.0000 1.3865 0.6000 0.8319 2.0000 0.4531 83.0000 3.7169 0.6000 2.2302 2.0000 0.8060 9 4.0000 8.9384 0.6000 5.36302.0000 3.0048 10 5.0000 23.9479 0.6000 14.3687 2.0000 4.0567 11 1.00000.0 0.6000 0.0 3.0000 0.0 12 2.0000 0.0 0.6000 0.0 3.0000 0.0 13 3.00000.0 0.6000 0.0 3.0000 0.0 14 4.0000 0.1293 0.6000 0.0776 3.0000 0.129315 5.0000 2.2007 0.6000 1.3204 3.0000 1.0634wherein:

BST is Braak Stage

ME1T4 is the extracellular tangle countPC is an estimate of the PHF-tau concentration per cell (calculated asabove)PT4 is the PHF content ascribed to extracellular tangles (PC x MEIT4)REG3B is the grouping of brain regions into 3 groups as per FIGS. 26 and27 of the SE1T4 is the standard error of the extracellular tangle count

Example 1 Aggregated Tau in Braak Staging

Based on immunochemical properties (Refs 26, 27, 30), it is possible todistinguish intracellular tangles from extracellular tangles. Bothfrequency of cases with tangles in these categories (ie probability) andtheir quantity (ie counts per mm²) were determined in a prospective caseseries and grouped into the regions known to represent stages in theprogression of pathology according to the system of Braak and Braak

As shown in FIG. 25, stages 2-4 can be clearly distinguished from stage1 on the basis of probability of extracellular tangles in E2/Trans andE4/HC. Also shown are the figures for F/T/P regions (neocorticalregions—frontal, temporal, parietal).

Conversely, intracellular tangles provide a poor basis fordiscrimination of early stages in these regions, but a good basis fordiscriminating stages 4 and 5 using neocortical regions. Similarly, whencases with MMSE scores greater than 21 in the 12 months prior to deathwere selected, similar results were obtained. Again, similar resultswere obtained when tangle densities were determined.

These results can be converted into approximations for the quantity ofaggregated tau protein in the extracellular space expressed in μg/g ofbrain tissue as described in Materials and Methods above.

The results are shown in Table 1. These are underestimates, as thetangle counts underestimate the quantity of aggregated tau protein.

TABLE 1 ESTIMATE PHF-TAU CONTENT IN ECT'S BY REGION AND STAGE REGION BSTPHF/TAU (μg/g) E2/TRANS 1 0.62 2 10.49 3 23.34 4 52.46 5 69.12 HC/E4 1 02 0.83 3 2.23 4 5.36 5 14.36 F/T/P 1 0 2 0 3 0 4 0.08 5 1.32

Table 1 shows quantity of aggregated tau protein in the extracellularspace expressed in μg/g of brain tissue as a function of Braak staging.The data was calculated as described in the materials and methods.

In summary, these results demonstrate that extracellular deposits ofPHF-tau in medial temporal lobe structures provide a basis for empiricalstaging of the neurofibrillary degeneration of AD. Such staging couldonly be accomplished by radio-imaging methods provided suitable ligandscould be created.

Example 2 Assessment of Compounds Binding within the Aggregated RepeatDomain of PHF-Core Tau Protein

A prototype compound was obtained as one component of the crude,commercially-available preparation of thioflavin-S was separated into˜20 distinct constituents by analytical thin-layer chromatography, andpreparative chromatography. Tests showed that not all of theconstituents were able to act as effective tangle ligands. Specifically,pure primulin (FIG. 5, compound 1a) was found to label tangles, but thebenzothiazole thioflavin-T (FIG. 5, compound 1b) was much lesseffective, although it labelled amyloid preferentially.

Furthermore, compound 1a was found to displace compound 1b at tangleswhen the latter was introduced at 10-fold excess into crude tangleextracts.

A possible difference was postulated to be the sulphonate group atposition 1 (FIG. 5, compound 2 [2-(4-amino phenyl)-6-methyl-1-sulphonatebenzothiazole]). However, primulin (compound 1a) was found to displacethis from tangles (though not amyloid). Therefore, tangle labelling isnot due solely to the sulphonated benzothiazole structure, indicatingthat a longer aromatic structure is required.

Purified thiazin red (FIG. 5, compound 3a) was found to compete withprimulin at equivalent concentrations, whereas the compound 3b (thiazinyellow, FIG. 5) did not. Therefore, an extended aromatic benzothiazolestructure does not, per se, determine high binding affinity withintangles.

In order to define a minimum critical requirement for competitivebinding, the sulphonated benzothiazole was extended by addition of asingle phenyl group across a diamino-linkage. This compound (FIG. 4,compound 4a), although not fluorescent, was found to compete out thiazinred and primulin fluorescence at equivalent concentrations. Compound 4atherefore defines the minimum critical structure required for highaffinity binding within the tangle.

In order to prove that the binding site within the tangle was in factthe PHF itself, compound 4a was further extended with addition of abiotin group (FIG. 4, compound 4b). Since this was still found tocompete primulin and thiazin red, compound 4b preserved high affinitybinding within the tangle. Furthermore, immunogold-conjugatedanti-biotin antibody was found to label isolated PHFs pre-incubated withcompound 4b, whereas no labelling was demonstrated withoutpre-incubation or pre-incubation with biotin alone (FIG. 31 b). Finally,when a photo-activated conjugate of the compound was prepared, it waspossible to identify and sequence the labelled protein. This was foundto be the same core tau fragment as that isolated from the core of thePHF, which comprises the repeat region of the tau protein.

In summary, these results demonstrate unequivocally that the bindingsite for compounds 4a and 4b is within the aggregated repeat domain ofthe tau protein of the PHF-core. Furthermore, they demonstrate thatcompound 4a can be used as a chelate for addition of functional groupswithout disturbing ligand activity within the PHF core. Therefore,compound 4a could be used as chelate for addition of technetium or otherimaging moiety to generate a ligand suitable for detecting e.g.extracellular tangles in AD.

Example 3 Determination of Optimum Dimensions of Ligand Molecules

FIG. 14 shows three of the structures described above, along with theirdimensions as indicated. For example, the C11-C1 distance and C10-C1distance are shown for primulin, a benzothiazole analogue (denoted‘analog’), and ‘thiazin yellow’.

FIGS. 15 and 16 illustrate the crystal structures of the ‘B’ part of theprimulin structure (Soon-Beng Teo et al., 1995, Acta Crystallogr., Sect.C, 591. As can be seen from FIG. 16, which is a ‘side-on’ view, themolecule is essentially flat, although it has a slight twist. The' A′part of primulin can be computed from the same molecule. From this, onecan derive measures of A+B, which provide an indication of the actuallength of one of the active species of the present invention.

To compute the size of the “analog” shown in FIG. 14, measurement A wasused from the data of FIG. 15, and measurement B was determined from amolecule denoted N2A and shown in FIGS. 17 and 18 (Gilardi, R. D., 1972,Acta Chrystallogr., Sect. B, 107). As can be seen from the side-on viewin FIG. 18, this part of the molecule is completely flat. The samemeasurements apply to thiazin red, which is identical in its dimensionsto the “analog”.

The size of thiazin yellow (shown in FIG. 14) was determined as follows.The ‘A’ part comes from the molecule of FIG. 15 which was used forprimulin, while the ‘B’ part comes from the molecule shown in FIGS. 19and 20 (Gladkova et al., 1972, Kristallografiya 41). Again, part B ofthe molecule is completely flat, and the only difference with respect tothe molecule shown in FIG. 17 is the distance between the aromaticgroups.

FIGS. 21 and 22 illustrate how the molecule of FIG. 15 crystallises inspace. As can be seen, the molecule forms an alternating ‘herring-bone’pattern, and does not stack. In comparison, the crystal structure ofmethylene blue indicates that the molecules form stacks with alternatingsheets of water molecules between the pi-bonded stacks.

Table 2 tabulates the minimum, maximum and mean dimensions for primulin(“PRIM”), the analog (“ANAL”), thiazin yellow, and the benzothiazoleunit alone (i.e. structures 1b and 2 as shown in FIG. 5). Thecorresponding methylene blue dimensions are given as ‘MBCC’ (carbon tocarbon) and ‘MBNN’ (nitrogen to nitrogen):

1 2 3 4 5 6 PRIM ANAL THIAZY BENZTHIA MBCC MBNN Minimum 14.7830 15.050015.7270 8.7700 7.0849 9.9600 Maximum 15.1120 15.2610 16.1380 8.95507.4443 9.9600 Mean 14.9475 15.1680 15.9273 8.8625 7.3031 9.9600

FIG. 23 shows a comparison of the mean, maximum and minimum extents ofmolecules which are active ligands (primulin and “analog”), and thiazinyellow (which is inactive as a ligand). The dimensions are given inangstrom units (AU). In FIG. 24, a similar comparison is made for thebasic benzothiazole nucleus (i.e. molecules 1b and 2 of FIG. 5) and thediaminophenothiazines. These distances are carbon-to-carbon distances.

The above results illustrate that the molecules provided herein aresubstantially flat. There is, however, a fundamental difference inactivity between ligands according to the present invention and othermolecules discussed above. As is shown in the Figures, suitable ligandsaccording to the invention comprise long, flat molecules with dimensionsbetween 14.783 and 15.261 AU. On the other hand, a longer molecule, suchas thiazin yellow, which exceeds these dimensions (mean 15.927 AU) doesnot serve as an effective ligand, even though it is flat. However,certain shorter, flat, molecules bind preferentially to amyloid.

Example 4 PET Using Ligand Molecules and Inhibitors

FIGS. 11 to 13 indicate typical synthesis methods which could be used toconvert either the diaminophenothiazines or the “analog” into positronemitting species.

FIG. 11 b in particular shows a method whereby thionin is treated withNaH followed by labelled methyl iodide to give methylene blue. A similarprocedure can be adopted for the synthesis of methylene blue startingfrom Azure A and Azure B. Other strong bases may also be used.

Other methods that may be used include HCl and labelled MeOH; labelledtrimethyl phosphoric acid; labelled dimethylsulphoxide and Labelledformaldehyde. The chemistry of the syntheses and general methodology areall familiar to persons skilled in the art. These examples are givenwithout any implied restriction as to ultimate methodology.

Example 5 Blocking Ligands

Compounds such as thioflavin-T and -S strongly stain amyloid deposits.However FIG. 31 demonstrates that such compounds can be displaced fromtangles by primulin. Therefore these compounds may be used as blockingreagents to saturate binding sites which are not of interest withoutinhibiting the binding of ligands to aggregated tau.

Example 6 Comparison of Ligand Molecules and Inhibitors

There appears to be a fundamental difference in activity of themolecules which are effective ligands, compared with those which areeffective inhibitors of tau-tau binding. The benzothiazole molecule doesnot disrupt PHFs, nor indeed do any of the ligands, whereas thediaminophenothiazine series constitute PHF-disrupters and tauaggregation inhibitors.

Further investigations into the relationship betweenaggregation-dependent tau ligands and tau aggregation inhibitors werecarried out using primulin. Primulin in solution has a fluorescence peakat 520 nm. This shifts to 470 nm when primulin is bound within a purepreparation of PHFs (FIG. 6). Treatment of PHFs with citraconicanhydride, which has been shown to disrupt the structure of the PHF andliberate free tau (as well as reversing the charge on lysines), wasfound abolish the 470 nm fluorescence peak (FIG. 7). Therefore, bindingby such compounds is dependent on the polymerised state of tau found inthe PHF, but is not present in free tau.

Compounds have been identified which disrupt the structure of the PHFand reverse the proteolytic stability of the PHF core (see WO 96/30766).Examples of such compounds are shown in accompanying FIG. 8. The presentinventors have now identified that these compounds bind to tau at aspecific binding site within the high affinity tau-tau binding domain.However, it is found that such compounds may not disrupt the binding ofprimulin to tau in aggregated tau molecules, as shown by the retentionof the fluorescence peak of primulin at 470 nm in the presence of alcianblue (FIG. 9).

Thus it appears that although alcian blue can inhibit tau-tauinteractions, either the site of the inhibition, or perhaps the order ofbinding interaction at which it acts, are such as to leave binding sitefor SB-ligands extant. Thus compounds which act as ligands of aggregatedtau do not appear to bind at the same site(s) as compounds which aretau-aggregation inhibitors, although they may still affect theinhibitory properties of those inhibitors (see Example 7 below).

This point was further examined by studying the potency of typicalaggregated tau ligands as tau-aggregation inhibitors. It has been shownpreviously that tau aggregation inhibitors (e.g. diaminophenothiazines)can be identified on the basis of inhibition of tau-tau binding in asolid-phase assay (WO 96/30766). When tested in the same assay, primulinand thiazin red were found to be weak inhibitors of tau-tau binding(FIG. 10). Thus, although these compounds are potent ligands for tauwithin the PHF-core, they are at most weak inhibitors at the siterequired for inhibition of tau-tau binding.

Demonstration that compounds of the diaminophenothiazine-like class bindtau in the aggregated state is provided by the direct demonstration ofdisruption of PHF structure in the presence of sufficiently highconcentrations, particularly of compounds such as methylene blue. Thus,compounds of the diaminophenothiazine-like class which are inhibitors oftau-tau binding can serve as aggregated-tau ligands at lowerconcentrations.

In summary, the inventors have found that it is possible to define twoclasses of binding site within the core-PHF tau aggregate. Both arepotentially useful for the development of radiological imaging ligands:

(i) Sulphonated benzothiazole-like sites: compounds of this type,associated with suitable chelates such as technetium, may serve asligands for extracellular tangles, due to their size and charge.(ii) Diaminophenothiazine-like sites: such compounds, when suitablylabelled with a positron-emitting functional group, would serve asligands for all tau aggregates, and would be capable of crossing theblood-brain-barrier (Ref 36) and entering cells. Thus these compounds,and derivatives thereof, have potential use in the labelling ofintracellular tangles, e.g. those present in the brains of AD patients,or intracellular tangles when used at lower concentration.

Example 7 Example Assays for Identifying Further Diagnostic LigandsBased on Relief of Inhibition

(i) Preparation of Tau Protein in which Partial Aggregation hasOccurred.

The preparation (“Preparation 2”) is shown schematically in FIG. 32, anddiffers from earlier described methods (e.g. in WO96/30766—“Preparation1”—shown in (ii) below).

The recombinant cDNA plasmids are those described in WO96/30766 thedisclosure of which is herein incorporated by reference.

Briefly, tau cDNA was generated using standard protocols (Sambrook,Fritsch & Maniatis “Molecular cloning. A Laboratory Manual” (1989) ColdSpring Harbor Laboratory, N.Y.) from mRNA isolated from brain tissue ofan Alzheimer patient whose tissue was obtained 3 h after death. The cDNAlibrary was screened with synthetic 17-mer oligonucleotide probesderived from the sequence from part of a PHF core protein (Goedert etal. (1988) Proc. Natl. Acad. Sci. USA, 85, 4051-4055). Full length cDNAclones were subcloned into the EcoRI site of M13mp19 and site-directedmutagenesis used to introduce a NdeI site in the context of theinitiator codon. Following cleavage with NdeI and EcoRI, the resultingcDNA fragments were subcloned downstream of the T7 RNA polymerasepromoter into NdeI/EcoRI-cut expression plasmid pRK172 (McLeod et al.(1987) EMBO J., 6, 729-736). PRK172 is a derivative of pBR322 that ispropagated at very high copy number in E. coli due to removal of thepBR322 copy number control region. The plasmid carries an ampicillinresistance gene for selection of recombinant clones.

cDNA constructs coding for truncated forms of tau were prepared frommRNA as described in Novak et al. (1993) EMBO J., 12, 365-370. The mRNAwas used as a template for polymerase chain reaction (PCR) usingspecific oligonucleotide primers. The sense primer contained an NdeIsite and the anti-sense, an EcoRI site. PCR fragments were subclonedinto pRK172 as described above. The primers used for construction ofdGAE are given in FIG. 22. The authenticity of all DNA fragments usedfor expression was confirmed by full length sequencing of both strands.

Details for the construction of htau40 (“T40”) cDNA are described in(Goedert et al. (1989), Neuron 3: 519-526). This sequence is the largestform of tau found in the CNS and encodes tau protein that contains boththe 2 N-terminal inserts of 29 amino acids each and an extra 31 aminoacid repeat in the tubulin-binding domain. The DNA sequence and itspredicted amino acid sequence are shown in FIG. 21 (SEQ ID NO: 4).

Recombinant plasmids were used to transform E. coli BL21 (DE3) a strainused for prokaryotic expression which carries a chromosomal copy of thebacteriophage T7 RNA polymerase gene under control of the lac UV5promoter (Studier and Moffat (1986), J. Mol. Biol. 189, 113-130).Exponentially growing cultures were induced with IPTG (iso-propylthiogalactodise) for 3 h.

Large-scale purification (1 litre bacterial culture) of tau fragmentswas carried out as described by Goedert and Jakes (1990, EMBO J., 9,4225-4230), with minor modifications. Cells were disrupted by rapidfreezing of the cell pellet in liquid nitrogen. The pellets were thensuspended in buffer containing 50 mM PIPES, 1 mM dithiothreitol (DTT)(pH 6.8). The thermostable proteins in the supernatant were dialysedagainst PIPES/DTT, then applied to a column containing phosphocelluloseequilibrated in the same buffer. Tau protein was eluted with a gradientof NaCl (0-0.5M) in the above buffer. Fractions were analysed bySDS-PAGE and both Coomassie staining and immunoblotting. Those fractionscontaining tau were pooled, dialysed against 25 mM MES, 1 mM DTT (pH6.25) and stored at −20° C. at approximately 5 mg/ml. Proteinconcentrations were measured by the Lowry method (Harrington C R (1990),“Lowry protein assay containing sodium dodecyl sulphate in microtitreplates for protein determinations on fractions from brain tissue”,Analytical Biochemistry 186: 285-287).

Preparation 2 differs from Preparation 1 above in the followingrespects: (1) The concentration of cells at the sonication stage isincreased by 5-fold. (2) A batchwise adsorption of non-tau proteins toDE52 is included. (3) The proteins are not subjected to heat treatment.(4) The final step involves concentration using polyethylene glycol.

Escherichia coli is grown in 2×TY medium (Oxoid) supplemented withampicillin (50 micrograms per ml) to late-logarithmic phase. Cells from5 litres of culture are harvested by centrifugation and the cell pelletsfrozen rapidly over liquid nitrogen. The pellets are taken up with 50 mMPIPES (pH 6.8) containing 1 mM EDTA, 1 mM dithiothreitol and 1 mMphenylmethylsulphonylfluoride (PMSF) and bacteria lysed by sonication(2×3 min) at 4 C. The mixture is centrifuged at 10,000 rpm for 20 min.The supernatant is rotated with 1 gram of Whatman DE52 for 3 hr at 4 C.The mixture is separated on a column and the flow-through material thatfails to bind to DE52 is incubated for 3 hr at 4 C, with rotation, with0.4 g of Whatman P11 (freshly regenerated according to manufacturer'srecommendations). The column is washed with column buffer (50 mM PIPES,pH 6.8 containing 1 mM EGTA, 5 mM EDTA, 0.2 mM MgCl₂, 5 mM(3-mercaptoethanol and 1 mM PMSF). Tau protein is eluted stepwise with a0.1 to 1 M gradient of KCl in column buffer. Fractions containing tau(determined by immunoassay) are pooled and dialysed against 80 mM PIPES(pH6.8) containing 1 mM EGTA, 1 mM MgCl₂ and 5 mM β-mercatoethanol,using dialysis tubing with a molecular weight cut-off of 1,000. Thedialysate is concentrated by applying polyethylene glycol 8000 to theoutside of the sac for 2-3 hr. The final concentration of tau rangedfrom 3 to 10 mg per ml.

In a typical large scale preparative run, the specific immunoreactivityof tau is purified approximately 30- to 40-fold from the material thatfails to bind to DE52. Approximately 60% of the tau is recovered in thefinal product with 10% failing to bind to P11 and the remainder infractions ignored from the column.

Table 3 shows the details of a preparative run for dGA.“Purification-fold” is expressed as the ratio of specificimmunoreactivity for each fraction (ie immunoreactivity/proteinconcentration) to specific immunoreactivity in the DE flow-through.

FIG. 33 is a graphical plot of the data from Table 3.

Table 4 summarises yields from typical preparative runs for the tauprotein species: dGA, dGAE and hT40.

Tau Protein concentration (mg/ml) based on: Immunoreactivity Proteinyield preparation abs at 280 nm Protein assay (BSA ref) (mAb 7/51;AU/ml) (per 10 litres) dGAE (711) 3.3 3.8 300,000 100 mg dGA (1511) 8.18.4 600,000 122 mg T40 (1311) 3.5 3.1 125,000 120 mg based on extinctioncoefficients abs at 280 nm BSA (reference) 10 mg/ml 6.6 Tau protein 10mg/ml 14.7

Proteins were separated by running down a 50×1 cm sepharose CL-6B gelfiltration column equilibrated with PBS buffer and run at roomtemperature. A molecular weight standard curve was prepared for thecolumn by running molecular weight markers over the range 12,400 to200,000 down the column. A standard curve was prepared by plotting thelog₁₀ of the Mr in Kd against Ve/Vo for each protein standard, where Veis the elution volume for the standard and Vo is the void volume for thecolumn determined with blue dextran.

T40 or dGAE were loaded in 0.5 ml buffer containing 5% glycerol andcollected in 1 ml fractions. Presence of protein in the fractions wasdetermined spectrophotometrically by the absorbance at 280 nm. Presenceof dGAE or T40 was detected by ELISA with monoclonal antibody 7/51 or499 respectively. ELISA assay was carried out in 96 well PVC plates asfollows: 50 μl samples of each fraction incubate 1 hr at 37 C, washplate in 0.05% tween-20 and then block binding sites with 200 μl PBS+2%non fat milk powder for 1 hr at 37 C. Wash plate in 0.05% tween-20,incubate with 50 μl primary antibody diluted 1:10 in PBS+2% non fat milkpowder for 1 hr at 37 C. Wash plate in 0.05% tween-20, incubate with 50μl secondary antibody (goat anti-mouse IgG:HRP conjugate) diluted inPBS+2% non fat milk powder for 1 hr at 37 C. Wash plate in 0.05%tween-20 then rinse in deionised water, add 50 μl freshly preparedsubstrate (TMB [tetramethylbenzidine] in sodium acetate buffer pH5.0with H₂O₂, freshly prepared) and read rate of change in OD 650 over 2minutes.

The elution profile of purified dGAE and purified T40 is shown in FIGS.34 (dGAE) and 35 (hT40). Although both these fragments typically run atabout 12 kD and 55 kD respectively, about 64% of mAb 7.51immunoreactivity (dGAE) elutes in fractions corresponding to species ofsize>15 kD and about 50% of mAb 499 immunoreactivity (hT40) eluted infractions corresponding to species of size>60 kD. Thus the tau proteinsare present, at least in part, in pre-aggregated form.

(ii) Effect of Tau Aggregation Inhibitors Measured Using DifferentPreparations of Tau Protein

When the proteins dGA and dGAE were prepared as indicated above(“Preparation 2”), the properties of the tau-tau binding assay werealtered relative to the properties obtained using the preparative methoddescribed in WO96/30766 (“Preparation 1”).

The assay is performed using 96 well PVC plates (Falcon Cat. No. 353912are used), and the following steps:

1. 50 μl dGA (−10 pg/ml) in carbonate buffer, incubate 1 h at 37° C.(Carbonate buffer: 50 mM carbonate bicarbonate, pH 9.6 (Na₂CO₃ 1.59 g/l,NaHCO₃ 2.93 g/l))2. Wash plate in 0.05% Tween-20.3 200 μl PBS+2% Marvel, incubate 1 h at 37° C.4. Rinse plate 2× in deionised water, then wash in 0.05% Tween-20.5 50 μl dGAE (˜10 μg/ml) plus drug in PBS+1% fish skin gelatin+0.05%Tween-20, incubate 1 h at 37° C.6 Wash plate in 0.05% Tween-20.7 50 μl antibody 423 (1:10 dilution in PBS+2% Marvel), incubate 1 h at37° C.8 Rinse plate 2× in deionised water, then wash in 0.05% Tween-20.9 50 μl HRP-anti-mouse (1:1000 dilution in PBS+0.05% Tween-20), incubate1 h at 37° C.10 Wash plate in 0.05% Tween-20, then rinse 1× with deionised water.11 50 μl substrate solution, read immediately initial rate over 2 min inplate reader at OD650.(Substrate solution: 50 mM sodium acetate, pH 5.0+TMB (1 ml/100 ml of a10 mg/ml solution in DMSO)+H₂O₂ (10 μl/100 ml)).

The compounds thionine and tolonium chloride were found to requirehigher concentrations to exert inhibitory effects in Preparation 2 thanin Preparation 1. This is shown in FIGS. 36 and 37.

Furthermore, the compound dimethyl methylene blue (DMMB) was found tohave a higher inhibitory potency in Preparation 2 than in Preparation 1.This is shown in FIG. 38.

As similar differences could be seen in Tau protein prepared by thePreparation 1 method, but which had been allowed to aggregate in vitroover time, the interpretation of this effect is as follows. Higherconcentrations of compounds such as thionine and tolonium chloride arerequired since two effects have to be achieved in order to achievemaximal inhibition of tau-tau binding:

1. disruption of pre-existing aggregates in the aqueous-phase;2. inhibition of binding of aqueous-phase species to the solid phase.

The greater potency of DMMB in the Preparation 2 assay can be explainedby greater binding affinity at the site of action required for bothinhibitory effects.

Full-length tau protein (hT40) prepared according to the Preparation 2protocol demonstrated minimal tau-tau binding activity when used in theaqueous-phase with dGA in the solid-phase. However, when hT40 was usedin the solid-phase, binding of dGAE was similar to that obtained forbinding of dGAE to dGA in the solid-phase (see FIG. 39 a-c). Theinterpretation is that in hT40 aggregates formed in the aqueous-phasetau-tau binding has already occurred in or through domains required forthe binding interaction with dGA in the solid-phase. When hT40 is firstplated in the solid-phase, the binding to PVC unfolds theprotein/aggregates in such as manner as to make the critical tau-taubinding sites available.

(iii) Effect of Tau Aggregation Inhibitors Measured Using DifferentPreparations of Tau Protein

In the Preparation 2 assay format, potent ligands of the kind typifiedby Primulin and Thiazin Red have no inhibitory activity on tau-taubinding. This is shown in FIG. 40 (cf. FIG. 10). Indeed in this assay,these compounds enhance tau-tau binding at concentrations greater than100 μM (i.e. 100-fold molar ratio with respect to tau protein).

DMMB typically reduces tau-tau binding (with tau present typically at 1μM) to 23% at DMMB 5 μM and 17% at DMMB 15 μM relative to that seen inthe absence of DMMB.

Unexpectedly, this inhibitory effect can be completely reversed byco-incubation in the presence of increasing concentrations of highaffinity ligands typified by Primulin and Thiazin Red. This is shown inFIG. 41.

Therefore, aggregated-tau ligands may be characterised functionally ascompounds which do not themselves inhibit tau-tau binding, but block theinhibitory effects of potent inhibitors of tau-tau binding.

As can be seen in FIG. 42, inhibition of Tau-Tau binding produced byDMMB is progressively attenuated and reversed in the presence ofincreasing molar excess of Primulin. A similar effect can be shown forThiazin Red. This indicates that the maximum inhibitory effect of DMMBis reduced by these compounds, and hence that they are acting asnon-competitive inhibitors of DMMB. One possible explanation might bethat the ligands stabilise the tau aggregates used in the assay, forexample in regions outside the critical binding domain required for DMMBactivity, and hence prevent the inhibitory effect of DMMB on Tau-Taubinding.

FIGS. 43-45 show that for any given concentration of DMMB, there isquantitative enhancement of Tau-Tau binding in the presence of Primulin(43, 44) or Thiazin Red (45) which can be modelled by a standardMichaelis-Menten equation. This implies that the Tau-aggregationenhancement effect of these ligands is proportional to the fraction ofligand-binding sites occupied, presumably within the Tau aggregatesintroduced into the aqueous-phase of the assay. The mean Bmax for bothligands is ˜1.6. That is, the maximum ligand effect is to produce1.6-fold the Tau-Tau binding signal seen in the absence of any drug. Themean Kd for this effect is ˜15×. That is, for any given concentration ofDMMB>4 μM, 50% of maximal enhancement of Tau-Tau binding can be seenwhen the ligand molar excess is 15-fold relative to the concentration ofDMMB.

Example 8 Example Assays for Identifying Further Diagnostic LigandsBased on Ligands Provided Herein

Having defined two classes of ligands as described above suitable forlabelling PHFs in AD, further ligands can be developed using thecompounds/derivatives in screening assays. Furthermore, modellingmethods can be based on the ligands already presented.

(i) Identification of Novel Ligands at the Sulphonated-BenzothiazoleSite.

Using a suitably labelled preparation of a known sulphonatedbenzothiazole, incubated with a preparation of aggregated tau molecules(e.g. preaggregated tau in solution, or bound to a solid phase, orhighly enriched PHFs isolated from AD brain—see WO96/30766) compoundssuspected of being suitable ligands can be introduced, and theircapacity to compete with the known ligand in such a way as to preventbinding within the PHF can be tested.

(ii) Identification of Novel Ligands at the Phenothiazine Site.

The tau-tau binding assay described in WO 96/30766 can be used as apreliminary screen to identify potential inhibitors at the tau-taubinding site. Likewise, a suitably-labelled preparation of knowndiaminophenothiazines, incubated with a aggregated tau as describedabove, could be used to screen for other compounds which are suspectedof being competitors at this PHF-binding site and thus potentiallysuitable PHF ligands.

The physical implementation of competitive assays is well known in theart. It may include measurement of fluorescence, radioactivity or anyother suitable reporting system which derives from sulphonatedbenzothiazole-like compounds or diaminophenothiazine-like compounds notbound to PHFs, i.e. those which remain in solution.

REFERENCES

-   1 DeToleda-Morrell, L. et al. (1997), Neurobiology of Aging 18, 5,    463-8;-   2 De Leon et al. (1997), Neurobiol. Of Aging, 18, 1, 1-11;-   3 Mori, E et al. (1997), Am. J. Psychiatry 154:1, p 18;-   4 Juottonen, K. (1998); J. Neurol. Neurosurg. Psychiatry 65,    322-327;-   5 Bobinski, M. et al. (1999), Lancet 353, p. 38;-   6 Fox, N. C. (1999) Neurol. 52, 1687-9;-   7 Jack, C. R. et al. (1997) Neurol. 49: 786-794;-   8 Fox, N. et al. (1996), Brain 119, 2001-7;-   9 Johnson, K. A. et al. (1998), Neurol. 50, 1563-1571;-   10 Perez-Tur, J. et al. (1999), Neurol. 53, 411-3;-   11 Lehtovirta, M. et al. (1998) J. Neurol. Neurosurg. Psychiatry 64,    742-6;-   12 Nagy, Zs et al. (1999), Dement. Geriatr. Cogn. Disord. 10,    109-114;-   13 Ishii, K. et al. (1998), Neurol. 51, 125-130;-   14 Imamura, T et al. (1997), Neurosci. Lett. 235, 49-52;-   15 Minoshima, S. et al. (1997), Ann. Aurol. 42, 85-94;-   16 Ibanez, V. et al. (1998), Neurol. 50, 1585-1593;-   17 Wischik, C. W. et al. (2000) “Neurobiology of Alzheimer's    Disease”, Eds. Dawbarn et al., The Molecular and Cellular    Neurobiology Series, Bios Scientific Publishers, Oxford).-   18 Carretero, M. T. et al. (1995), Dementia 6, 281-5;-   19 Villareal, D. T. et al. (1998), Alzheimer's Dis. Rev. 3, 142-152;-   20 Marin, D. B. et al. (1998), Artherosclerosis 140, 173-180;-   21 Kuller, L. H. et al. (1998), Stroke 29, 388-398;-   22 Vargha-Khadem, F. et al. (1997), Science 277, p 376;-   23 Willingham, D. B. (1997), Neuron 18, 5-8;-   24 Lakmache, Y. et al. (1995), PNAS USA 95, 9042-6;-   25 Hodges, J. R. et al. (1999), PNAS USA 96, 9444-8;-   26 Mena, R. et al. (1995), Acta Neuropathol. 89, 50-6;-   27 Mena, R. et al. (1996), Acta Neuropathol. 91, 633-641;-   28 (deleted)-   29 Lai, R. et al. (1995) Neurobiol. Aging 16, 3, 433-445;-   30 Bondareff, W. et al. (1994) J. Neuropathol. Exp. Neurol. 53, 2,    158-164;-   31 Resch, J. F. et al. (1991) Bioorg. Med. Chem. Lett. 1, 10,    519-522;-   32 Novak, M. et al. (1993), EMBO J. 12, 1, 365-370;-   33 Wischik, C. W. et al. (1996), PNAS USA 93, 11213-8;-   34 Wischik C. W. et al. (1989), Curr. Opin. Cell Biol. 1, 115-122;-   35 WO 96/30766;-   36 Muller, T. (1992), Acta Anat. 144, 39-44.

1. A method for determining the stage of neurofibrillary degenerationassociated with a tauopathy in a subject believed to suffer from thedisease, which method comprises the steps of: (i) introducing into thesubject a ligand capable of labelling aggregated paired helical filament(PHF) tau protein, (ii) determining the presence and\or amount of ligandbound to extracellular aggregated PHF tau in the medial temporal lobe ofthe brain of the subject, (iii) correlating the result of thedetermination made in (ii) with the extent of neurofibrillarydegeneration in the subject.
 2. A method as claimed in claim 1 whereinthe determination in step (ii) is used to establish the density ligandbinding.
 3. A method as claimed in claim 1 or claim 2 wherein thecorrelation in step (iii) is made by reference to historical data.
 4. Amethod as claimed in any one of the preceding claims wherein thetauopathy is Alzheimer Disease (AD).
 5. A method as claimed in claim 4wherein the extent of neurofibrillary degeneration is related to theneuropathological staging of the progression of AD according to thedefined hierarchical system shown in FIG. 2 c.
 6. A method as claimed inany one of the preceding claims wherein the ligand is capable ofcrossing the blood brain barrier.
 7. a method as claimed in any one ofthe preceding claims wherein the ligand is conjugated, chelated, orotherwise associated, with a detectable chemical group.
 8. A method asclaimed in claim 7 wherein the ligand is labelled for SPECT and is notcapable taken up intracellularly.
 9. A method as claimed in claim 8wherein a ligand comprises a technetium-chelating group.
 10. A method asclaimed in claim 7 wherein the ligand is labelled for positron emissiontomography (PET).
 11. A method as claimed in claim 10 wherein the ligandcomprises a positron-emitting carbon, optionally incorporated into amethyl group present in the ligand.
 12. A method as claimed in any oneof the preceding claims wherein the ligand is a compound of the formula:

wherein: W is S, O, or NH; exactly one of X, Y, and Z is CH or N; theothers of X, Y, and Z are CH; M¹ is an alkali metal cation selectedfrom: Li, Na, K, or Cs. RL is a rigid linker group; Ar¹ is an C₅₋₂₀arylgroup; n is an integer from 0 to 3; and, each R^(BT) is independently acore substituent.
 13. A method as claimed in claim 12 wherein each of X,Y, and Z is CH.
 14. A method as claimed in claim 13 wherein the ligandis a compound of the formula:

wherein: M¹ is an alkali metal cation selected from: Li, Na, K, or Cs.RL is a rigid linker group; Ar¹ is an C₅₋₂₀aryl group; n is an integerfrom 0 to 3; and, each R^(BT) is independently a benzothiazolesubstituent.
 15. A method as claimed in any one of claims 12 to 14wherein each of the rigid linker group, RL, and the aryl group, Ar¹, aresubstantially planar.
 16. A method as claimed in any one of claims 12 to15 wherein the rigid linker group, RL, and the aryl group, Ar¹, togetherwith the core group, form a compound which is substantially planar. 17.A method as claimed in any one of claims 12 to 16 wherein the twist isno greater than that of the compound of FIG.
 16. 18. A method as claimedin any one of claims 12 to 17 wherein the compound has a compound lengthwhich is from about 14.7 AU to about 15.3 AU.
 19. A method as claimed inany one of claims 12 to 18 wherein each R^(BT) is independentlyC₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, or amino.
 20. Amethod as claimed in claim 19 wherein each R^(BT) is independently -Me,-Et, -nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl,—Br, —I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂,N(iPr)₂, or —N(nPr)₂.
 21. A method as claimed in claims 19 wherein eachR^(BT) is independently C₁₋₄alkyl.
 22. A method as claimed in any one ofclaims 19 to 21 wherein n is 1, and R^(BT) is independently -Me, -Et,-nPr, or -iPr.
 23. A method as claimed in any one of claims 12 to 22wherein the ligand has the following formula:


24. A method as claimed in claim 23 wherein the ligand has the followingformula:


25. A method as claimed in any one of claims 12 to 24 wherein RL is agroup of the formula:

wherein: m is an integer from 0 to 4, and each R^(RL) is independently arigid linker aryl substituent, and the ligand has the formula:


26. A method as claimed in claim 25 wherein each R^(RL) is independentlyC₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, or amino.
 27. Amethod as claimed in claim 26 wherein each R^(RL) is independently -Me,-Et, -nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl,—Br, —I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂,N(iPr)₂, or —N(nPr)₂.
 28. A method as claimed in claim 26 wherein eachR^(RL) is independently C₁₋₄alkyl.
 29. A method as claimed in any one ofclaims 1 to 24 wherein RL is a group of the formula:


30. A method as claimed in any one of claims 1 to 24 wherein RL is agroup of the formula:

wherein p is an integer from 0 to 3, and each R^(RL) is independently arigid linker aryl substituent, and the compounds have the formula:


31. A method as claimed in claim 30 wherein each R^(RL) is independentlyC₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, or amino.
 32. Amethod as claimed in claim 31 wherein each R^(RL) is independently -Me,-Et, -nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl,—Br, —I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂,N(iPr)₂, or —N(nPr)₂.
 33. A method as claimed in claim 31 wherein eachR^(RL) is independently C₁₋₄ alkyl.
 34. A method as claimed in any oneof claims 12 to 24 and 30 to 33 wherein RL is a group of the formula:


35. A method as claimed in any one of claims 12 to 34 wherein Ar¹ isselected from groups derived from benzene (C₆), naphthalene (C₁₀),anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene(C₁₆).
 36. A method as claimed in any one of claims 12 to 34 wherein Ar¹is selected from: C₅heteroaryl groups derived from furan (oxole),thiophene (thiole), pyrrole (azole), imidazole (1,3-diazole), pyrazole(1,2-diazole), triazole, oxazole, isoxazole, thiazole, isothiazole,oxadiazole, and oxatriazole; and C₆heteroaryl groups derived fromisoxazine, pyridine (azine), pyridazine (1,2-diazine), pyrimidine(1,3-diazine), pyrazine (1,4-diazine), triazine, tetrazole, andoxadiazole (furazan).
 37. A method as claimed in any one of claims 12 to34 wherein Ar¹ is selected from: C₉heterocyclic groups derived frombenzofuran, isobenzofuran, indole, isoindole, purine, benzimidazole;C₁₀heterocyclic groups derived from quinoline, isoquinoline,benzodiazine, pyridopyridine, quinoxaline; C₁₃heterocyclic groupsderived from carbazole; and, C₁₄heterocyclic groups derived fromacridine, xanthene, phenoxathiin, phenazine, phenoxazine, phenothiazine.38. A method as claimed in claim 35 wherein Ar¹ is an aryl group havinga phenyl core, and has the formula:

wherein q is an integer from 0 to 5; and each R^(A) is independently anaryl substituent; R^(C), if present, is a reactive conjugatingsubstituent, or R^(C) is, or contains, a detectable label; and thecompound has the formula:


39. A method as claimed in claim 38 wherein R^(C) is present and is areactive conjugating substituent, and is, or contains, a reactivefunctional group suitable for conjugation to another molecule bychemical reaction therewith, to form a covalent linkage therebetween.40. A method as claimed in claim 39 wherein R^(C) is present and is, orcontains, an active ester.
 41. A method as claimed in claim 40 whereinR^(C) is present and is, or contains, a succinimidyl ester.
 42. A methodas claimed in claim 39 wherein R^(C) is present and is a reactiveconjugating substituent, and is, or contains, a moiety suitable forconjugation to another molecule by a strong non-covalent interaction.43. A method as claimed in claim 42 wherein R^(C) is present and is, orcontains, biotin.
 44. A method as claimed in claim 39 wherein R^(C) ispresent and is a reactive conjugating substituent, and is, or contains,a moiety suitable for conjugation to another molecule by complex orchelate formation.
 45. A method as claimed in claim 44 wherein R^(C) ispresent and is, or contains, a technetium-chelating group.
 46. A methodas claimed in claim 45 wherein R^(C) is present and is, or containsdiethylenetriaminepentaacetic acid.
 47. A method as claimed in claim 38wherein R^(C) is present and is, or contains, a detectable label.
 48. Amethod as claimed in claim 47 wherein R^(C) is present and is, orcontains, a dye, a fluorescent marker, an antigenic group, a stable oran unstable isotope, or a positron-emitting carbon atom.
 49. A method asclaimed in claim 48 wherein R^(C) is present and is, or contains, ¹⁸F.50. A method as claimed in claim 48 wherein R^(C) is present and is, orcontains, a positron-emitting carbon atom.
 51. A method as claimed inany one of claims 38 to 50 wherein each R^(A) is independently selectedfrom: —OH, —NH₂, —NHR¹, —NR¹R², —SO₃M², and C₁₋₄alkyl; wherein: R¹ andR² are each C₁₋₄alkyl, and M² is an alkali metal cation selected fromLi, Na, K, or Cs.
 52. A method as claimed in claim 51 wherein at leastone R^(A) is —OH or —NH₂.
 53. A method as claimed in claim 52 whereinAr¹ is an aryl group having an amino-substituted phenyl core, and hasthe formula:

wherein r is an integer from 0 to 4, and each R^(A) is independently anaryl substituent.
 54. A method as claimed claim 53 wherein each R^(A) isindependently selected from: —OH, —NH₂, —NHR¹, —NR¹R², —SO₃M², andC₁₋₄alkyl; wherein: R¹ and R² are each C₁₋₄alkyl, and M² is an alkalimetal cation selected from Li, Na, K, or Cs.
 55. A method as claimed inclaim 54 wherein the ligand has the formula:


56. A method as claimed in claim 55 wherein the ligand has the formula:


57. A method as claimed in claim 52 wherein Ar¹ is an aryl group havinga hydroxy-substituted phenyl core, and has the formula:

wherein s is an integer from 0 to 4, and each R^(A) is independently anaryl substituent, and R^(C), if present, is a reactive conjugatingsubstituent, or R^(C) is, or contains, a detectable label.
 58. A methodas claimed in claim 57 wherein the ligand is as defined in any one ofclaims 108 to
 150. 59. A method as claimed in claim 35 wherein Ar¹ is anaryl group having a naphthyl core, and has the formula:

wherein t is an integer from 0 to 3, u is an integer from 0 to 4, andeach R^(A) is independently an aryl substituent, and the compound hasthe formula:


60. A method as claimed in claim 59 wherein Ar¹ is an aryl group havinga hydroxy-substituted naphthyl core, and has the formula:

wherein v is an integer from 0 to 2, u is an integer from 0 to 4, andeach R^(A) is independently an aryl substituent.
 61. A method as claimedin claim 59 or claim 60 wherein each R^(A) is independently selectedfrom: —OH, —NH₂, —NHR¹, —NR¹R², —SO₃M², and C₁₋₄alkyl; wherein: R¹ andR² are each C₁₋₄alkyl, and M² is an alkali metal cation selected from:Li, Na, K, or Cs.
 62. A method as claimed in claim 61 wherein the ligandhas the formula:


63. A method as claimed in claim 62 wherein the ligand has the formula:


64. A method as claimed in any one of claims 1 to 11 wherein the ligandis a compound of one of the following formulae:

wherein: each of R₁, R₃, R₄, R₆, R₇ and R₉ is independently hydrogen,halogen, hydroxy, carboxy, substituted or unsubstituted alkyl,haloalkyl, or alkoxy; R₅ is independently hydrogen, hydroxy, carboxy,substituted or unsubstituted alkyl, haloalkyl, or alkoxy; R₁₀ and R₁₁are independently selected from hydrogen, hydroxy, carboxy, substitutedor unsubstituted alkyl, haloalkyl, or alkoxy; or a pharmaceuticallyacceptable salt thereof.
 65. A method as claimed in claim 64 wherein:each of R₁, R₃, R₄, R₆, R₇ and R₉ is independently hydrogen, halogen,hydroxy, carboxy, substituted or unsubstituted C₁₋₆alkyl, C₁₋₄haloalkyl,or C₁₋₆alkoxy; R₅ is independently hydrogen, hydroxy, carboxy,substituted or unsubstituted C₁₋₆alkyl, C₁₋₄haloalkyl, or C₁₋₆alkoxy;R₁₀ and R₁₁ are independently selected from hydrogen, hydroxy, carboxy,substituted or unsubstituted C₁₋₆alkyl, C₁₋₄haloalkyl, or C₁₋₆alkoxy.66. A method as claimed in claim 65 wherein said C₁₋₆alkyl is selectedfrom: methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, and isohexyl.
 67. Amethod as claimed in claim 65 or claim 66 wherein the substituents ofsaid substituted C₁₋₆alkyl are selected from: mercapto, thioether,nitro, amino, aryloxy, halogen, hydroxyl, carbonyl, C₅₋₂₀aryl,C₁₋₆cycloalkyl, and non-aryl C₃₋₂₀heterocyclyl.
 68. A method as claimedin any one of claims 65 to 67 wherein said C₁₋₄haloalkyl is selectedfrom: chloromethyl, 2-bromethyl, 1-chloroisopropyl, 3-fluoropropyl,2,3-dibrombutyl, 3-chloroisobutyl, iodo-t-butyl, and trifluoromethyl.69. A method as claimed in any one of claims 64 to 68 wherein the ligandis an acid addition salt formed between a compound described in saidclaims and an acid.
 70. A method as claimed in claim 69 wherein the acidis an inorganic acid or an organic acid.
 71. A method as claimed inclaim 70 wherein the ligand is a chloride salt.
 72. A method as claimedin claim 70 wherein said organic acid is selected from: acetic acid,citric acid, maleic acid, fumaric acid, tartaric acid, methanesulphonicacid, and p-toluenesulphonic acid.
 73. A method as claimed in any one ofclaims 64 to 72 wherein: R₁, R₃, R₄, R₆, R₇ and R₉ are independently —H,—CH₃, —C₂H₅, or —C₃H₇; R₁₀ and R₁₁ are independently —H, —CH₃, —C₂H₅ or—C₃H₇; R₅ is independently —H, —CH₃, —C₂H₅, or —C₃H₇.
 74. A method asclaimed in claim 73 wherein the ligand is shown in FIG. 8 b.
 75. Amethod as claimed in any one of claims 64 to 74 wherein the ligandcomprises a positron-emitting carbon.
 76. A method as claimed in any oneof the preceding claims wherein (ii) further comprises the step ofadditionally determining the presence and\or amount of a ligand bound tointracellular aggregated tau in a neocortical structure of the brain ofthe subject.
 77. A method as claimed in claim 76 wherein the ligand usedto bind to extracellular aggregated PHF tau in the medial temporal lobeand the ligand used to bind to intracellular aggregated PHF tau in theneocortical structure of the brain are labelled distinctively.
 78. Amethod as claimed in claim 76 or claim 77 wherein the ligand used tobind intracellular aggregated tau is the ligand described in any one ofclaims 64 to
 75. 79. A method as claimed in any one of the precedingclaims wherein the ligand binds aggregated PHF tau preferentially withrespect to competing binding sites present in the relevant region of thebrain.
 80. A method as claimed in any one of the preceding claimswherein steps (i) and\or (ii) of the method are performed in conjunctionwith the further step of introducing into the subject a further blockingligand which labels the competing binding sites present in the relevantregion of the brain preferentially to the ligand used to bind aggregatedPHF tau.
 81. A method as claimed in claim 80 wherein the blocking ligandis [¹⁸]FDDNP.
 82. A method as claimed in claim 80 wherein the blockingligand is a benzthiazole of the formula:

wherein: n is an integer from 0 to 4; each R^(BT) is independently ablocking ligand benzothiazole substituent; m is an integer from 0 to 4;each R^(P) is independently a phenylene substituent; each R isindependently —H or an amino substituent; and, either: R^(N) and X⁻ areboth absent and the associated (tertiary) nitrogen atom is neutral; or:R^(N) is a benzothiazolino substituent and the associated (quaternary)nitrogen atom bears a positive charge, and X⁻ is a counter ion.
 83. Amethod as claimed in claim 82 wherein the blocking ligand isthioflavin-T.
 84. A method as claimed in claim 82 wherein each R^(BT),is independently C₁₋₄alkyl, —SO₃H, or —SO₃M³, wherein M³ is a cation.85. A method as claimed in claim 84 wherein M³ is an alkali metal cationselected from: Li, Na, K, or Cs.
 86. A method as claimed in any one ofclaims 82 to 85 wherein n is 1, and R^(BT) is -Me, -Et, -nPr, or -iPr.87. A method as claimed in claim 86 wherein n is 1, and R^(BT) is -Me.88. A method as claimed in claim 84 wherein one of the R^(BT) groups is—SO₃H or —SO₃M³, and another of the R^(BT) groups is C₁₋₄alkyl.
 89. Amethod as claimed in any one of claims 82 to 85 wherein n is 2, and oneR^(BT) is C₁₋₄alkyl, and one R^(BT) is —SO₃H or —SO₃M³.
 90. A method asclaimed in claim 89 wherein n is 2, and one R^(BT) is -Me, and oneR^(BT) is —SO₃H or —SO₃M³.
 91. A method as claimed in any one of claims82 to 90 wherein R^(N) and X⁻ are both absent and the associated(tertiary) nitrogen atom is neutral.
 92. A method as claimed in any oneof claims 82 to 90 wherein R^(N) is a benzothiazolino substituent andthe associated (quaternary) nitrogen atom bears a positive charge, andX⁻ is a counter ion.
 93. A method as claimed in any one of claims 82 to90 wherein R^(N) is C₁₋₄ alkyl.
 94. A method as claimed in claim 93wherein R^(N) is -Me, -Et, -nPr, or -iPr.
 95. A method as claimed in anyone of claims 82 to 90 wherein X⁻ is Cl⁻, Br⁻, and I⁻.
 96. A method asclaimed in any one of claims 82 to 95 wherein R^(P) is C₁₋₄alkyl.
 97. Amethod as claimed in any one of claims 82 to 96 wherein each R is —H,and the amino group is —NH₂.
 98. A method as claimed in any one ofclaims 82 to 96 wherein one R is —H and one R is an amino substituent.99. A method as claimed in any one of claims 82 to 96 wherein each R isan amino substituent.
 100. A method as claimed in claim 99 wherein eachof the amino substituents is C₁₋₄alkyl.
 101. A method as claimed in anyone of claims 82 to 96 wherein the amino group, —NR₂, is —NH₂, —NHMe,—NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂, N(iPr)₂, or —N(nPr)₂.
 102. Amethod as claimed in claim 101 wherein the blocking ligand is abenzthiazole of the formula:


103. A method as claimed in claim 101 wherein the blocking ligand is abenzthiazole of the formula:


104. A method as claimed in any one of the preceding claims for use inthe diagnosis or prognosis of a tauopathy in a subject believed tosuffer from said disease.
 105. A method as claimed in claim 104 whereintauopathy is AD.
 106. A ligand as described in any one of claims 1 to 79for use in a method of diagnosis or prognosis of a tauopathy in asubject believed to suffer from said disease, said method being a methodas described in claim 104 or claim
 105. 107. A ligand as described inany one of claims 1 to 79 for use in a method for determining theeffectiveness of a treatment of a subject with a tau-tau aggregationinhibitor to inhibit neurofibrillary degeneration in that subject, themethod comprising use of a method as described in any one of claims 1 to103.
 108. A ligand of the formula:

wherein: M¹ is an alkali metal cation; n is an integer from 0 to 3; eachR^(BT) is a independently benzothiazole substituent; m is an integerfrom 0 to 4; each R^(RL) is independently a rigid linker arylsubstituent; s is an integer from 0 to 4; each R^(A) is independently anaryl substituent; and, R^(C), if present, is a reactive conjugatingsubstituent, or R^(C) is, or contains, a detectable label.
 109. A ligandas claimed in claim 108 wherein M¹ is Li, Na, K, or Cs.
 110. A ligand asclaimed in claim 108 or claim 109 wherein each R^(BT) is independentlyC₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano, halo, or amino.
 111. Aligand as claimed in claim 110 wherein each R^(BT) is independently -Me,-Et, -nPr, -iPr, —OH, —OMe, —OEt, —O(nPr), —O(iPr), —NO₂, —CN, —F, —Cl,—Br, —I, —NH₂, —NH₂, —NHMe, —NHEt, —NH(iPr), —NH(nPr), —NMe₂, —NEt₂,N(iPr)₂, or —N(nPr)₂.
 112. A ligand as claimed in claim 110 wherein eachR^(BT) is independently C₁₋₄alkyl.
 113. A ligand as claimed in claim 112wherein each R^(BT) is independently -Me, -Et, -nPr, or -iPr.
 114. Aligand as claimed in claim 113 wherein each R^(BT) is -Me.
 115. A ligandas claimed in claim 113 wherein n is 1, and R^(BT) is independently -Me,-Et, -nPr, or -iPr.
 116. A ligand as claimed in claim 115 wherein n is1, and R^(BT) is -Me.
 117. A ligand as claimed in any one of claims 108to 116 wherein the benzothiazole group has the following formula:


118. A ligand as claimed in claim 117 wherein the ligand has thefollowing formula:


119. A ligand as claimed in any one of claims 108 to 118 wherein eachR^(RL) is independently C₁₋₄alkyl, hydroxy, C₁₋₄alkoxy, nitro, cyano,halo, or amino.
 120. A ligand as claimed in claim 119 wherein eachR^(RL) is independently -Me, -Et, -nPr, -iPr, —OH, —OMe, —OEt, —O(nPr),—O(iPr), —NO₂, —CN, —F, —Cl, —Br, —I, —NH₂, —NH₂, —NHMe, —NHEt,—NH(iPr), —NH(nPr), —NMe₂, —NEt₂, N(iPr)₂, or —N(nPr)₂.
 121. A ligand asclaimed in claim 120 wherein each R^(RL) is independently C₁₋₄alkyl.122. A ligand as claimed in any one of claims 108 to 121 wherein eachR^(A) is independently selected from: —OH, —NH₂, —NHR¹, —NR¹R², —SO₃M²,and C₁₋₄alkyl; wherein: R¹ and R² are each C₁₋₄alkyl, and M² is analkali metal cation.
 123. A ligand as claimed in claim 122 wherein M² isLi, Na, K, or Cs.
 124. A ligand as claimed in any one of claims 108 to123 wherein R^(C) is present and is a reactive conjugating substituent,and is, or contains, a reactive functional group suitable forconjugation to another molecule by chemical reaction therewith, to forma covalent linkage therebetween.
 125. A ligand as claimed in claim 124wherein R^(C) is present and is, or contains, an active ester.
 126. Aligand as claimed in claim 125 wherein R^(C) is present and is, orcontains, a succinimidyl ester.
 127. A ligand as claimed in claim 124wherein R^(C) is present and is a reactive conjugating substituent, andis, or contains, a moiety suitable for conjugation to another moleculeby a strong non-covalent interaction.
 128. A ligand as claimed in claim127 wherein R^(C) is present and is, or contains, biotin.
 129. A ligandas claimed in claim 124 wherein R^(C) is present and is a reactiveconjugating substituent, and is, or contains, a moiety suitable forconjugation to another molecule by complex or chelate formation.
 130. Aligand as claimed in claim 129 wherein R^(C) is present and is, orcontains, a technetium-chelating group.
 131. A ligand as claimed inclaim 130 wherein R^(C) is present and is, or containsdiethylenetriaminepentaacetic acid.
 132. A ligand as claimed in any oneof claims 108 to 123 wherein R^(C) is present and is, or contains, adetectable label.
 133. A ligand as claimed in claim 132 wherein R^(C) ispresent and is, or contains, a dye, a fluorescent marker, an antigenicgroup, a stable or an unstable isotope, or a positron-emitting carbonatom.
 134. A ligand as claimed in claim 133 wherein R^(C) is present andis, or contains, ¹⁸F.
 135. A ligand as claimed in claim 134 whereinR^(C) is present and is, or contains, a positron-emitting carbon atom.136. A ligand as claimed in any one of claims 108 to 135 wherein theligand has the formula:


137. A ligand as claimed in claim 136 wherein the ligand has theformula:


138. A ligand as claimed in claim 137 wherein the ligand has theformula:


139. A ligand as claimed in claim 137 wherein the ligand has theformula:


140. A ligand as claimed in claim 139 wherein the ligand has theformula:


141. A ligand as claimed in claim 140 wherein the ligand has theformula:


142. A ligand as claimed in claim 137 wherein the ligand has theformula:


143. A ligand as claimed in claim 142 wherein the ligand has theformula:


144. A ligand as claimed in claim 143 wherein the ligand has theformula:


145. A ligand as claimed in claim 142 wherein the ligand has theformula:


146. A ligand as claimed in claim 145 wherein the ligand has theformula:


147. A ligand as claimed in claim 145 wherein the ligand has theformula:


148. A ligand as claimed in any one of claim 147 wherein the ligand hasthe formula:


149. A ligand as claimed in claim 148 wherein the ligand has theformula:


150. A ligand as claimed in any one of claims 136 to 149 which isconjugated, chelated, or otherwise associated, with a detectablechemical group.
 151. A diagnostic composition comprising as its activeingredient at least 90%, 95%, or 98% of the ligand of any one of claims108 to
 150. 152. A diagnostic composition as claimed in claim 151further comprising a carrier material or other pharmaceutically- andphysiologically-acceptable excipient.
 153. A method of labellingaggregated tau or tau-like molecules, comprising contacting theaggregated molecules with a ligand or composition of any one of claims108 to
 152. 154. An in vitro method of diagnosis or prognosis of atauopathy in a subject believed to suffer from the disease, the methodcomprising (i) obtaining a sample of appropriate tissue from a subject;(ii) contacting the sample with a ligand or composition of any one ofclaims 108 to 152; (iii) detecting the amount and\or localisation of theligand bound to the sample (iv) correlating the result of (iii) with thestage or severity of the disease in the subject.
 155. A method asclaimed in claim 153 where the tauopathy is AD.
 156. A ligand accordingto any one of claims 108 to 150 for use in a method of diagnosis,prognosis or therapeutic treatment of the human or animal body for atauopathy.
 157. A ligand as claimed in claim 156 where the tauopathy isAD.
 158. A method for identifying a compound capable of bindingaggregated tau protein, the method comprising the steps: (i) providing asample of aggregated tau protein to which a ligand as claimed in any oneof claims 108 to 150 has been bound, (ii) contacting the sample with theputative tau-binding compound, (iii) determining the extent ofdisplacement of the ligand from the aggregated tau protein by theputative tau-binding compound (iv) correlating the result of thedetermination made in (iii) with the ability of the compound to bindaggregated tau protein.
 159. A method as claimed in claim 158 whereinthe aggregated tau protein is in solution or bound to a solid phase.160. An in vitro method for identifying a ligand capable of labelingaggregated PHF tau protein, the method comprising the steps of: (i)providing a first agent suspected of being capable of labelingaggregated PHF tau protein, (ii) contacting (a) a tau protein or aderivative thereof containing the tau core fragment bound to a solidphase so as to expose a high affinity tau capture site, with (b) aliquid phase tau protein or derivative thereof capable of binding to thesolid phase tau protein or derivative, and (c) said selected first agentand (d) a second agent known to be tau-tau binding inhibitor, (iii)selecting first agent which fully or partially relieves the inhibitionof binding of the liquid phase tau protein or derivative of (b) to thesolid phase tau protein or derivative of (a) by the inhibitor (d). 161.A method as claimed in claim 160 wherein step (ii) is carried out inconjunction with: (ibis) contacting (a) a tau protein or a derivativethereof containing the tau core fragment bound to a solid phase so as toexpose a high affinity tau capture site, with (b) a liquid phase tauprotein or derivative thereof capable of binding to the solid phase tauprotein or derivative, and (c) said first agent, and (ibis.1) detectinginhibition of tau-tau binding as exhibited by inhibition of binding ofthe liquid phase tau protein or derivative of (b) to the solid phase tauprotein or derivative of (a), (ibis.2) selecting first agent which hasminimal or absent activity as tau-tau binding inhibitors and\oroptionally enhance tau-tau binding.
 162. A method as claimed in claim160 or claim 161 wherein the inhibitor is a diaminophenathiozine asdescribed in any one of claims 64 to
 74. 163. A method as claimed inclaim 162 wherein the inhibitor is DMMB.
 164. A method as claimed in anyone of claims 160 to 163 wherein the liquid phase tau protein orderivative is prepared in a form which has undergone partial aggregationprior to exposure to the solid phase.
 165. A method as claimed in claim174 wherein said preparation of liquid phase tau protein or derivativecomprises the steps of: (i) sonicating said tau protein or derivativeand\or (ii) exposing said tau protein or derivative to PEG through asemi-permeable membrane.
 166. A method as claimed in any one of claims160 to 165, wherein the step of contacting said agent and liquid phasetau protein or derivative with said solid phase tau protein orderivative is carried out in an alkaline buffer of pH 9-10.
 167. Amethod as claimed in any one of claims 160 to 165, wherein the step ofcontacting said agent and liquid phase tau protein or derivative withsaid solid phase tau protein or derivative is carried out inphysiological buffer conditions.
 168. A method as claimed in any one ofclaims 160 to 165, wherein the step of contacting the or each agent andliquid phase tau protein or derivative with said solid phase tau proteinor derivative is carried out in 50-400 mM sodium chloride or a salt orsalt mixture of comparable ionic strength, and in a pH range of 4-10.169. A method as claimed in any one of claims 160 to 168, wherein saidliquid phase tau protein or derivative is labeled.
 170. A method asclaimed in any one of claims 160 to 168 wherein said liquid phase tauprotein or derivative is immunologically distinguishable from said solidphase tau protein or derivative, and the binding is detected byantibodies.
 171. A method as claimed in any one of claims 160 to 170wherein a truncated tau protein corresponding to the core fragment andterminating at Ala390 (dGA) is plated on a solid phase in bufferconditions unfavourable to tau-tau association,
 172. A method as claimedin any one of claims 160 to 171 wherein a truncated tau proteincorresponding to the core fragment and terminating at Glue-391 (dGAE) ora full-length protein is added in liquid phase together with the or eachagent
 173. A method as claimed in any one of claims 158 to 172 furthercomprising the step of formulating the agent as a diagnostic orprognostic reagent.
 174. A method as claimed in any one of claims 158 to172 further comprising the step of using the agent as a ligand in themethod of any one of claims 1 to 11.